Dna-pk inhibitors

ABSTRACT

The present invention relates to compounds useful as inhibitors of DNA-PK. The invention also provides pharmaceutically acceptable compositions comprising said compounds and methods of using the compositions in the treatment of various diseases, conditions, or disorders.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.U.S. 62/618,598, filed Jan. 17, 2018, which is incorporated by referenceherein in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. The ASCII copy, created on Jan. 15, 2019, isnamed 14390-686 Sequence listing_ST25.txt and is 8 KB in size.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to compounds useful as inhibitors ofDNA-dependent protein kinase (DNA-PK). The invention also providespharmaceutically acceptable compositions comprising the compounds of theinvention and methods of using the compositions in the treatment ofcancer, and for increasing genome editing efficiency by administering aDNA-PK inhibitor and a genome editing system to a cell(s).

BACKGROUND OF THE INVENTION

Ionizing radiation (IR) induces a variety of DNA damage of which doublestrand breaks (DSBs) are the most cytotoxic. These DSBs can lead to celldeath via apoptosis and/or mitotic catastrophe if not rapidly andcompletely repaired. In addition to IR, certain chemotherapeutic agentsincluding topoisomerase II inhibitors, bleomycin, and doxorubicin alsocause DSBs. These DNA lesions trigger a complex set of signals throughthe DNA damage response network that function to repair the damaged DNAand maintain cell viability and genomic stability. In mammalian cells,the predominant repair pathway for DSBs is the Non-Homologous EndJoining Pathway (NHEJ). This pathway functions regardless of the phaseof the cell cycle and does not require a template to re-ligate thebroken DNA ends. NHEJ requires coordination of many proteins andsignaling pathways. The core NHEJ machinery consists of the Ku70/80heterodimer and the catalytic subunit of DNA-dependent protein kinase(DNA-PKcs or DNA-PK), which together comprise the active DNA-PK enzymecomplex. DNA-PKcs is a member of the phosphatidylinositol3-kinase-related kinase (PIKK) family of serine/threonine proteinkinases that also includes ataxia telangiectasia mutated (ATM), ataxiatelangiectasia and Rad3-related (ATR), mTOR, and four PI3K isoforms.However, while DNA-PKcs is in the same protein kinase family as ATM andATR, these latter kinases function to repair DNA damage through theHomologous Recombination (HR) pathway and are restricted to the S and G2phases of the cell cycle. While ATM is also recruited to sites of DSBs,ATR is recruited to sites of single stranded DNA breaks.

NHEJ is thought to proceed through three key steps: recognition of theDSBs, DNA processing to remove non-ligatable ends or other forms ofdamage at the termini, and finally ligation of the DNA ends. Recognitionof the DSB is carried out by binding of the Ku heterodimer to the raggedDNA ends followed by recruitment of two molecules of DNA-PKcs toadjacent sides of the DSB; this serves to protect the broken terminiuntil additional processing enzymes are recruited. Recent data supportsthe hypothesis that DNA-PKcs phosphorylates the processing enzyme,Artemis, as well as itself to prepare the DNA ends for additionalprocessing. In some cases DNA polymerase may be required to synthesizenew ends prior to the ligation step. The auto-phosphorylation ofDNA-PKcs is believed to induce a conformational change that opens thecentral DNA binding cavity, releases DNA-PKcs from DNA, and facilitatesthe ultimate religation of the DNA ends.

It has been known for some time that DNA-PK^(−/−) mice arehypersensitive to the effects of IR and that some non-selective smallmolecule inhibitors of DNA-PKcs can radiosensitize a variety of tumorcell types across a broad set of genetic backgrounds. While it isexpected that inhibition of DNA-PK will radiosensitize normal cells tosome extent, this has been observed to a lesser degree than with tumorcells likely due to the fact that tumor cells possess higher basallevels of endogenous replication stress and DNA damage (oncogene-inducedreplication stress) and DNA repair mechanisms are less efficient intumor cells. Most importantly, an improved therapeutic window withgreater sparing of normal tissue will be imparted from the combinationof a DNA-PK inhibitor with recent advances in precision delivery offocused IR, including image-guide RT (IGRT) and intensity-modulated RT(IMRT).

Inhibition of DNA-PK activity induces effects in both cycling andnon-cycling cells. This is highly significant since the majority ofcells in a solid tumor are not actively replicating at any given moment,which limits the efficacy of many agents targeting the cell cycle.Equally intriguing are recent reports that suggest a strong connectionbetween inhibition of the NHEJ pathway and the ability to killtraditionally radioresistant cancer stem cells (CSCs). It has been shownin some tumor cells that DSBs in dormant CSCs predominantly activate DNArepair through the NHEJ pathway; it is believed that CSCs are usually inthe quiescent phase of the cell cycle. This may explain why half ofcancer patients may experience local or distant tumor relapse despitetreatment as current strategies are not able to effectively target CSCs.A DNA-PK inhibitor may have the ability to sensitize these potentialmetastatic progenitor cells to the effects of IR and select DSB-inducingchemotherapeutic agents.

Given the involvement of DNA-PK in DNA repair processes, an applicationof specific DNA-PK inhibitory drugs would be to act as agents that willenhance the efficacy of both cancer chemotherapy and radiotherapy.Accordingly, it would be desirable to develop compounds useful asinhibitors of DNA-PK.

In addition, precise genome targeting technologies are needed to enablesystematic engineering of genetic variations. The use of genome editingsystems, specifically Clustered Regularly Interspaced Short PalindromicRepeats (CRISPR)-endonuclease based genome editing technology has grownexponentially in the past few years. The type II CRISPR-Cas9 bacterialinnate immune system has emerged as an effective genome editing tool fortargeted modification of the human genome (Wiedenheft, B. 2012; Hsu, P.D. eta. 2014). Recently, CRISPR-Cpf genome editing systems have beendescribed. CRISPR-endonuclease based genome editing is dependent, inpart, upon non-homologous end joining (NHEJ) and homology directedrepair (HDR) pathways to repair DNA double strand breaks. Cellularrepair mechanism favors NHEJ over HDR.

While the achievement of insertion or deletions (indels) from NHEJ is upto 70% effective in some reports, the efficiency of HDR remainschallenging, with rates at less than 1%.

Accordingly, a need exists for increasing genome editing efficiency, inparticular, HDR efficiency. Another application of specific DNA-PKinhibitory drugs would be to act as agents that will enhance theefficacy of genome editing systems.

SUMMARY OF THE INVENTION

It has been found that compounds of this invention, and pharmaceuticallyacceptable compositions thereof, are effective as inhibitors of DNA-PK.Accordingly, the invention features compounds having the generalformula:

or a pharmaceutically acceptable salt thereof, where each of R¹, R², X,Ring A, Ring B and Ring C is as defined elsewhere herein.

The invention also provides pharmaceutical compositions that include acompound of formula I and a pharmaceutically acceptable carrier,adjuvant, or vehicle. These compounds and pharmaceutical compositionsare useful for treating or lessening the severity of cancer.

The compounds and compositions provided by this invention are alsouseful for the study of DNA-PK in biological and pathological phenomena;the study of intracellular signal transduction pathways mediated by suchkinases; and the comparative evaluation of new kinase inhibitors.

The present invention can also improve HDR efficiency by suppressingNHEJ enzymes such as DNA-PK using DNA-PK inhibitors.

In some embodiments, the disclosure provides a method of editing one ormore target genomic regions, the method includes administering to one ormore cells that have one or more target genomic regions, a genomeediting system and a compound represented by Formula I:

or pharmaceutically acceptable salts thereof, where each of R¹, R², X,Ring A, Ring B and Ring C independently is as defined elsewhere herein.

In some embodiments, the disclosure also provides a method of repairinga DNA break in one or more target genomic regions via a homologydirected repair (HDR) pathway, the method includes administering to oneor more cells that have one or more target genomic regions, a genomeediting system and a compound represented by Formula I orpharmaceutically acceptable salts thereof.

The genome editing system interacts with a nucleic acid(s) of the targetgenomic regions, resulting in a DNA break, and wherein the DNA break isrepaired at least in part via a HDR pathway.

In some embodiments, the disclosure also provides a method of inhibitingor suppressing repair of a DNA break in one or more target genomicregions via a NHEJ pathway, the method includes administering to one ormore cells that have one or more target genomic regions, a genomeediting system and a compound represented by Formula I orpharmaceutically acceptable salts thereof.

The genome editing system interacts with a nucleic acid(s) of the one ormore target genomic regions, resulting in a DNA break, and whereinrepair of the DNA break via a NHEJ pathway is inhibited or suppressed.

In some embodiments, the disclosure also provides a method of modifyingexpression of one or more genes or proteins, the method includesadministering to one or more cells that comprise one or more targetgenomic regions, a genome editing system and a compound represented byFormula I or pharmaceutically acceptable salts thereof.

The genome editing system interacts with a nucleic acid(s) of the one ormore target genomic regions of a target gene(s), resulting in editingthe one or more target genomic regions and wherein the edit modifiesexpression of a downstream gene (s) and/or protein(s) associated withthe target gene(s).

In some embodiments, a kit or composition is provided for editing one ormore target genomic regions. In some embodiments, the kit or compositionincludes a genome editing system; and a compound represented by FormulaI or pharmaceutically acceptable salts thereof.

Other features, objects, and advantages of the invention are apparent inthe detailed description that follows. It should be understood, however,that the detailed description, while indicating embodiments and aspectsof the invention, is given by way of illustration only, not limitation.Various changes and modification within the scope of the invention willbecome apparent to those skilled in the art from the detaileddescription.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the design of the gene editing assays.

FIG. 2 is a graph showing gene editing rates in BECs treated with aDNA-PK inhibitor.

FIGS. 3A and 3B are graphs showing gene editing rates following DNA-PKinhibitor treatment in CD34⁺ cells from two different donors.

FIG. 4 is a graph showing gene editing rates in iPSCs treated with aDNA-PK inhibitor.

FIG. 5 is a graph showing gene editing kinetics in BECs at the DNA-PKinhibitor ECmax.

FIG. 6 is a graph showing gene editing kinetics in BECs at the DNA-PKinhibitor EC50.

FIG. 7 is a bar graph showing HDR rates for gene editing componentsdelivered by lipid-mediated transfection in BECs.

FIG. 8 is a graph showing gene editing rates in CD34⁺ treated with aDNA-PK inhibitor.

FIG. 9 is a graph showing gene editing rates in CD34⁺ treated with aDNA-PK inhibitor.

FIG. 10 is a graph showing gene editing rates in CD34⁺ treated with aDNA-PK inhibitor.

FIG. 11 depicts the design of a gene editing strategy to performHomology-Driven Repair (HDR) using CRISPR-Cas9 using AAV donors.

FIG. 12 is a graph showing precise gene editing by HDR mediated by AAVdonors, CRISPR-Cas9 and a selective DNA-PK inhibitor.

DETAILED DESCRIPTION OF THE INVENTION Definitions and GeneralTerminology

As used herein, the following definitions shall apply unless otherwiseindicated. For purposes of this invention, the chemical elements areidentified in accordance with the Periodic Table of the Elements, CASversion, and the Handbook of Chemistry and Physics, 75^(th) Ed. 1994.Additionally, general principles of organic chemistry are described in“Organic Chemistry,” Thomas Sorrell, University Science Books,Sausalito: 1999, and “March's Advanced Organic Chemistry,” 5^(th) Ed.,Smith, M. B. and March, J., eds. John Wiley & Sons, New York: 2001, theentire contents of which are hereby incorporated by reference.Generally, nomenclatures utilized in connection with, and techniques of,cell and tissue culture, molecular biology, and protein and oligo- orpolynucleotide chemistry and hybridization described herein are thosewell-known and commonly used in the art. Standard techniques are usedfor recombinant DNA, oligonucleotide synthesis, and tissue culture andtransformation (e.g., electroporation, lipofection). Enzymatic reactionsand purification techniques are performed according to manufacturer'sspecifications or as commonly accomplished in the art or as describedherein. The foregoing techniques and procedures are generally performedaccording to conventional methods well known in the art and as describedin various general and more specific references that are cited anddiscussed throughout this disclosure. See e.g., Sambrook et al.Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989)).

As described herein, compounds of the invention may optionally besubstituted with one or more substituents, such as are illustratedgenerally above, or as exemplified by particular classes, subclasses,and species of the invention. It will be appreciated that the phrase“optionally substituted” is used interchangeably with the phrase“substituted or unsubstituted.” In general, the term “substituted,”whether preceded by the term “optionally” or not, refers to thereplacement of one or more hydrogen radicals in a given structure withthe radical of a specified substituent. Unless otherwise indicated, anoptionally substituted group may have a substituent at eachsubstitutable position of the group. When more than one position in agiven structure can be substituted with more than one substituentselected from a specified group, the substituent may be either the sameor different at each position.

As described herein, when the term “optionally substituted” precedes alist, said term refers to all of the subsequent substitutable groups inthat list. For example, if X is halogen; optionally substituted C₁₋₃alkyl or phenyl; X may be either optionally substituted alkyl oroptionally substituted phenyl. Likewise, if the term “optionallysubstituted” follows a list, said term also refers to all of thesubstitutable groups in the prior list unless otherwise indicated. Forexample: if X is halogen, C₁₋₃ alkyl, or phenyl, wherein X is optionallysubstituted by J^(X), then both C₁₋₃ alkyl and phenyl may be optionallysubstituted by J^(X). As is apparent to one having ordinary skill in theart, groups such as H, halogen, NO₂, CN, NH₂, OH, or OCF₃ would not beincluded because they are not substitutable groups. As is also apparentto a skilled person, a heteroaryl or heterocyclic ring containing an NHgroup can be optionally substituted by replacing the hydrogen atom withthe substituent. If a substituent radical or structure is not identifiedor defined as “optionally substituted,” the substituent radical orstructure is unsubstituted.

Combinations of substituents envisioned by this invention are preferablythose that result in the formation of stable or chemically feasiblecompounds. The term “stable,” as used herein, refers to compounds thatare not substantially altered when subjected to conditions to allow fortheir production, detection, and, preferably, their recovery,purification, and use for one or more of the purposes disclosed herein.In some embodiments, a stable compound or chemically feasible compoundis one that is not substantially altered when kept at a temperature of40° C. or less, in the absence of moisture or other chemically reactiveconditions, for at least a week.

The term “alkyl” or “alkyl group,” as used herein, means astraight-chain (i.e., unbranched) or branched, substituted orunsubstituted hydrocarbon chain that is completely saturated. Unlessotherwise specified, alkyl groups contain 1-8 carbon atoms. In someembodiments, alkyl groups contain 1-6 carbon atoms, and in yet otherembodiments, alkyl groups contain 1-4 carbon atoms (represented as “C₁₋₄alkyl”). In other embodiments, alkyl groups are characterized as “C₀₋₄alkyl” representing either a covalent bond or a C₁₋₄ alkyl chain.Examples of alkyl groups include methyl, ethyl, propyl, butyl,isopropyl, isobutyl, sec-butyl, and tert-butyl. The term “alkylene,” asused herein, represents a saturated divalent straight or branched chainhydrocarbon group and is exemplified by methylene, ethylene,isopropylene and the like. The term “alkylidene,” as used herein,represents a divalent straight chain alkyl linking group. The term“alkenyl,” as used herein, represents monovalent straight or branchedchain hydrocarbon group containing one or more carbon-carbon doublebonds. The term “alkynyl,” as used herein, represents a monovalentstraight or branched chain hydrocarbon group containing one or morecarbon-carbon triple bonds.

The term “cycloalkyl” (or “carbocycle”) refers to a monocyclic C₃-C₈hydrocarbon or bicyclic C₈-C₁₂ hydrocarbon that is completely saturatedand has a single point of attachment to the rest of the molecule, andwherein any individual ring in said bicyclic ring system has 3-7members. Suitable cycloalkyl groups include, but are not limited to,cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.

The term “heterocycle,” “heterocyclyl,” “heterocycloalkyl,” or“heterocyclic” as used herein refers to a monocyclic, bicyclic, ortricyclic ring system in which at least one ring in the system containsone or more heteroatoms, which is the same or different, and that iscompletely saturated or that contains one or more units of unsaturation,but which is not aromatic, and that has a single point of attachment tothe rest of the molecule. In some embodiments, the “heterocycle,”“heterocyclyl,” “heterocycloalkyl,” or “heterocyclic” group has three tofourteen ring members in which one or more ring members is a heteroatomindependently selected from oxygen, sulfur, nitrogen, or phosphorus, andeach ring in the system contains 3 to 8 ring members.

Examples of heterocyclic rings include, but are not limited to, thefollowing monocycles: 2-tetrahydrofuranyl, 3-tetrahydrofuranyl,2-tetrahydrothiophenyl, 3-tetrahydrothiophenyl, 2-morpholino,3-morpholino, 4-morpholino, 2-thiomorpholino, 3-thiomorpholino,4-thiomorpholino, 1-pyrrolidinyl, 2-pyrrolidinyl, 3-pyrrolidinyl,1-tetrahydropiperazinyl, 2-tetrahydropiperazinyl,3-tetrahydropiperazinyl, 1-piperidinyl, 2-piperidinyl, 3-piperidinyl,1-pyrazolinyl, 3-pyrazolinyl, 4-pyrazolinyl, 5-pyrazolinyl,1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-piperidinyl,2-thiazolidinyl, 3-thiazolidinyl, 4-thiazolidinyl, 1-imidazolidinyl,2-imidazolidinyl, 4-imidazolidinyl, 5-imidazolidinyl; and the followingbicycles: 3-1H-benzimidazol-2-one, 3-(1-alkyl)-benzimidazol-2-one,indolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, benzothiolane,benzodithiane, and 1,3-dihydro-imidazol-2-one.

The term “heteroatom” means one or more of oxygen, sulfur, nitrogen, orphosphorus, including any oxidized form of nitrogen, sulfur, orphosphorus; the quaternized form of any basic nitrogen; or asubstitutable nitrogen of a heterocyclic ring, for example N (as in3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl) or NR⁺ (as inN-substituted pyrrolidinyl).

The term “unsaturated,” as used herein, means that a moiety has one ormore units of unsaturation.

The term “alkoxy,” or “thioalkyl,” as used herein, refers to an alkylgroup, as previously defined, attached to the principal carbon chainthrough an oxygen (“alkoxy”) or sulfur (“thioalkyl”) atom.

The terms “haloalkyl,” “haloalkenyl,” and “haloalkoxy” mean alkyl,alkenyl, or alkoxy, as the case may be, substituted with one or morehalogen atoms. The term “halogen” means F, Cl, Br, or I.

The term “aryl” used alone or as part of a larger moiety as in“aralkyl,” “aralkoxy,” or “aryloxyalkyl,” refers to a monocyclic,bicyclic, or tricyclic carbocyclic ring system having a total of six tofourteen ring members, wherein said ring system has a single point ofattachment to the rest of the molecule, at least one ring in the systemis aromatic and wherein each ring in the system contains 4 to 7 ringmembers. The term “aryl” may be used interchangeably with the term “arylring.” Examples of aryl rings include phenyl, naphthyl, and anthracene.

The term “heteroaryl,” used alone or as part of a larger moiety as in“heteroaralkyl,” or “heteroarylalkoxy,” refers to a monocyclic,bicyclic, and tricyclic ring system having a total of five to fourteenring members, wherein said ring system has a single point of attachmentto the rest of the molecule, at least one ring in the system isaromatic, at least one ring in the system contains one or moreheteroatoms independently selected from nitrogen, oxygen, sulfur orphosphorus, and wherein each ring in the system contains 4 to 7 ringmembers. The term “heteroaryl” may be used interchangeably with the term“heteroaryl ring” or the term “heteroaromatic.”

Further examples of heteroaryl rings include the following monocycles:2-furanyl, 3-furanyl, N-imidazolyl, 2-imidazolyl, 4-imidazolyl,5-imidazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-oxazolyl,4-oxazolyl, 5-oxazolyl, N-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 2-pyridyl,3-pyridyl, 4-pyridyl, 2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl,pyridazinyl (e.g., 3-pyridazinyl), 2-thiazolyl, 4-thiazolyl,5-thiazolyl, tetrazolyl (e.g., 5-tetrazolyl), triazolyl (e.g.,2-triazolyl and 5-triazolyl), 2-thienyl, 3-thienyl, pyrazolyl (e.g.,2-pyrazolyl), isothiazolyl, 1,2,3-oxadiazolyl, 1,2,5-oxadiazolyl,1,2,4-oxadiazolyl, 1,2,3-triazolyl, 1,2,3-thiadiazolyl,1,3,4-thiadiazolyl, 1,2,5-thiadiazolyl, pyrazinyl, 1,3,5-triazinyl, andthe following bicycles: benzimidazolyl, benzofuryl, benzothiophenyl,indolyl (e.g., 2-indolyl), purinyl, quinolinyl (e.g., 2-quinolinyl,3-quinolinyl, 4-quinolinyl), and isoquinolinyl (e.g., 1-isoquinolinyl,3-isoquinolinyl, or 4-isoquinolinyl).

As described herein, a bond drawn from a substituent to the center ofone ring within a multiple-ring system (as shown below) representssubstitution of the substituent at any substitutable position in any ofthe rings within the multiple ring system. For example, Structure arepresents possible substitution in any of the positions shown inStructure b.

This also applies to multiple ring systems fused to optional ringsystems (which would be represented by dotted lines). For example, inStructure c, X is an optional substituent both for ring A and ring B.

If, however, two rings in a multiple ring system each have differentsubstituents drawn from the center of each ring, then, unless otherwisespecified, each substituent only represents substitution on the ring towhich it is attached. For example, in Structure d, Y is an optionallysubstituent for ring A only, and X is an optional substituent for ring Bonly.

The term “protecting group,” as used herein, represent those groupsintended to protect a functional group, such as, for example, analcohol, amine, carboxyl, carbonyl, etc., against undesirable reactionsduring synthetic procedures. Commonly used protecting groups aredisclosed in Greene and Wuts, Protective Groups In Organic Synthesis,3^(rd) Edition (John Wiley & Sons, New York, 1999), which isincorporated herein by reference. Examples of nitrogen protecting groupsinclude acyl, aroyl, or carbamyl groups such as formyl, acetyl,propionyl, pivaloyl, t-butylacetyl, 2-chloroacetyl, 2-bromoacetyl,trifluoroacetyl, trichloroacetyl, phthalyl, o-nitrophenoxyacetyl,α-chlorobutyryl, benzoyl, 4-chlorobenzoyl, 4-bromobenzoyl,4-nitrobenzoyl and chiral auxiliaries such as protected or unprotectedD, L or D, L-amino acids such as alanine, leucine, phenylalanine and thelike; sulfonyl groups such as benzenesulfonyl, p-toluenesulfonyl and thelike; carbamate groups such as benzyloxycarbonyl,p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl,p-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl,p-bromobenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl,3,5-dimethoxybenzyloxycarbonyl, 2,4-dimethoxybenzyloxycarbonyl,4-methoxybenzyloxycarbonyl, 2-nitro-4,5-dimethoxybenzyloxycarbonyl,3,4,5-trimethoxybenzyloxycarbonyl,1-(p-biphenylyl)-1-methylethoxycarbonyl,α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, benzhydryloxycarbonyl,t-butyloxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl,ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl,2,2,2,-trichloroethoxycarbonyl, phenoxycarbonyl, 4-nitrophenoxycarbonyl, fluorenyl-9-methoxycarbonyl, cyclopentyloxycarbonyl,adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl and thelike, arylalkyl groups such as benzyl, triphenylmethyl, benzyloxymethyland the like and silyl groups such as trimethylsilyl and the like.Preferred N-protecting groups are formyl, acetyl, benzoyl, pivaloyl,t-butylacetyl, alanyl, phenylsulfonyl, benzyl, t-butyloxycarbonyl (Boc)and benzyloxycarbonyl (Cbz). Examples of hydroxyl protecting groupsinclude ethers, such as tetrahydropyranyl, tert butyl, benzyl, allyl,and the like; silyl ethers such as trimethyl silyl, triethyl silyl,triisopropylsilyl, tert-butyl diphenyl silyl, and the like; esters suchas acetyl, trifluoroacetyl, and the like; and carbonates. Hydroxylprotecting groups also include those appropriate for the protection ofphenols.

Unless otherwise depicted or stated, structures recited herein are meantto include all isomeric (e.g., enantiomeric, diastereomeric, andgeometric (or conformational)) forms of the structure; for example, theR and S configurations for each asymmetric center, (Z) and (E) doublebond isomers, and (Z) and (E) conformational isomers. Therefore, singlestereochemical isomers as well as enantiomeric, diastereomeric, andgeometric (or conformational) mixtures of the present compounds arewithin the scope of the invention. Compounds that have been drawn withstereochemical centers defined, usually through the use of a hatched (

) or bolded (

) bond, are stereochemically pure, but with the absolute stereochemistrystill undefined. Such compounds can have either the R or Sconfiguration. In those cases where the absolute configuration has beendetermined, the chiral center(s) are labeled (R) or (S) in the drawing.

Unless otherwise stated, all tautomeric forms of the compounds of theinvention are within the scope of the invention. Additionally, unlessotherwise stated, structures depicted herein are also meant to includecompounds that differ only in the presence of one or more isotopicallyenriched atoms. For example, compounds having the present structuresexcept for the replacement of hydrogen by deuterium or tritium, or thereplacement of a carbon by a ¹³C- or ¹⁴C-enriched carbon are within thescope of this invention. Such compounds are useful, for example, asanalytical tools, probes in biological assays, or as DNA-PK inhibitorswith an improved therapeutic profile.

Description of Compounds

In one aspect, the invention features compounds having the formula(III-E-1) or (III-E-2), or a pharmaceutically acceptable salt thereof,

X is O or NR; wherein R is H or C₁-C₄ alkyl.

Y is O, or NR; wherein R is H or C₁-C₄ alkyl.

R³ is hydrogen, C₁₋₄ alkyl, or OC₁₋₂ alkyl.

R¹ is a 6-membered heteroaromatic ring containing one or two nitrogenatoms wherein the heteroaromatic ring may be substituted by 0, 1, 2 or 3substituents R² independently selected from the group consisting ofhalo, CN, C₁-C₄-alkyl, C₁-C₄-haloalkyl, C₃-C₄-cycloalkyl, OR⁶, C(═O)OR⁶,C(═O)NR⁷R⁶, and NR⁴R⁵.

Each C₁-C₄-alkyl and C₁-C₄-haloalkyl is substituted by 0, 1, or 2 OR⁶groups.

Each R⁶ and R⁷ is independently H, C₁-C₄ alkyl or C₁-C₄-haloalkyl.

Each R⁴ and R⁵ is independently H, C₁-C₄ alkyl, or C(═O)C₁-C₄ alkyl.

R⁴ and R⁵ together with the N atom to which they are attached form aheterocyclic ring comprising 0 or 1 additional O or N atom wherein saidheterocyclic ring may be substituted by C₁C₄ alkyl or OR⁶.

Ring B is selected from the group consisting of:

wherein W is N or CR⁷; and Z is O or S; wherein R⁷ is H or C₁-C₄ alkyl.

In another aspect, R¹ is a 6-membered heteroaromatic ring containing oneor two nitrogen atoms wherein the heteroaromatic ring may be substitutedby 0, 1, or 2 substituents R² independently selected from the groupconsisting of C₁-C₄-alkyl, C₁-C₄-haloalkyl , C(═O)NHR⁶, and NR⁴R⁵.

R⁶ is C₁-C₄-alkyl.

each R⁴ and R⁵ is independently H, C₁-C₄-alkyl, or C(═O)C1-C4 alkyl.

R⁴ and R⁵ together with the N atom to which they are attached form aheterocyclic ring comprising 0 or 1 additional N atom wherein saidheterocyclic ring may be substituted by C₁-C₄-alkyl.

Ring B is selected from the group consisting of:

W is N or CR⁷; and Z is O or S; wherein R⁷ is H or C₁-C₄-alkyl.

In one embodiment, the compounds have the formula

In one embodiment, X is O.

In another embodiment, X is NH.

In one embodiment, Y is O.

In another embodiment, Y is NH.

In a further embodiment, X is O or NH, Y is O or NH, and R³ is hydrogen.

In another embodiment, X is O or NH, Y is O or NH, R³ is hydrogen and R¹is a pyrimidine ring which is substituted by 0, 1, or 2 substituents R²independently selected from the group consisting of C₁-C₄-alkyl,C₁-C₄-haloalkyl, C(═O)NHR²′, and NR⁴R⁵.

In another embodiment, X is O or NH, Y is O or NH, R³ is hydrogen and R¹is a pyrimidine ring which is substituted by one substituents R²selected from the group consisting of C₁-C₄-alkyl, C₁-C₄-haloalkyl,C(═O)NHR²′, and NR⁴R⁵.

In another embodiment, X is O or NH, Y is O or NH, R³ is hydrogen and R¹is a pyrimidine ring which is substituted by one substituent R² selectedfrom the group consisting of C₁-C₄-alkyl and C(═O)NHR²′.

In another embodiment, X is O or NH, Y is O or NH, R³ is hydrogen and R¹is a pyrimidine ring which is substituted by one substituent R² selectedfrom the group consisting of C₁-C₂-alkyl and C(═O)NHC₁-C₂-alkyl.

In another embodiment, X is O, Y is NH, R³ is hydrogen and R¹ is apyrimidine ring which is substituted by one substituent R² selected fromthe group consisting of C₁-C₂-alkyl and C(═O)NHC₁-C₂-alkyl.

In another embodiment, X is O or NH, Y is O or NH, R³ is hydrogen and R¹is a pyrimidine ring which is substituted by 0, 1, or 2 substituents R²independently selected from the group consisting of C₁-C₄-alkyl,C₁-C₄-haloalkyl, C(═O)NHR⁶, and NR⁴R⁵; Ring B is selected from the groupconsisting of:

W is N or CR³; and Z is O or S; wherein R³ is H or C₁-C₄ alkyl.

In another embodiment, X is O or NH, Y is O or NH, R³ is hydrogen and R¹is a pyrimidine ring which is substituted by 0, 1, or 2 substituents R²independently selected from the group consisting of C₁-C₄-alkyl,C₁-C₄-haloalkyl, C(═O)NHR⁶, and NR⁴R⁵; Ring B is

W is N or CR³; and Z is O or S; wherein R³ is H or C₁-C₄ alkyl.

In another embodiment, X is O or NH, Y is O or NH, R³ is hydrogen and R¹is a pyrimidine ring which is substituted by 0, 1, or 2 substituents R²independently selected from the group consisting of C₁-C₄-alkyl,C₁-C₄-haloalkyl, C(═O)NHR⁶, and NR⁴R⁵; Ring B is

W is N; and Z is O or S.

In another embodiment, X is O or NH, Y is O or NH, R³ is hydrogen and R¹is a pyrimidine ring which is substituted by 0, 1, or 2 substituents R²independently selected from the group consisting of C₁-C₄-alkyl andC(═O)NHR⁶; Ring B is

W is N; and Z is O or S.

In another embodiment, X is O or NH, Y is O or NH, R³ is hydrogen and R¹is a pyrimidine ring which is substituted by 0, 1, or 2 substituents R²independently selected from the group consisting of C₁-C₂-alkyl andC(═O)NH C₁-C₂-alkyl; Ring B is

W is N; and Z is O or S.

In another embodiment, the compounds have the formula

In another embodiment, X is NH.

In one embodiment, Y is O .

In another embodiment, Y is NH.

In a further embodiment, X is O or NH, Y is O or NH, and R³ is hydrogen.

In another embodiment, X is O or NH, Y is O or NH, R³ is hydrogen and R¹is a pyrimidine ring which is substituted by 0, 1, or 2 substituents R²independently selected from the group consisting of C₁-C₄-alkyl,C₁-C₄-haloalkyl, C(═O)NHR⁶, and NR⁴R⁵.

In another embodiment, X is O or NH, Y is O or NH, R³ is hydrogen and R¹is a pyrimidine ring which is substituted by one substituents R²selected from the group consisting of C₁-C₄-alkyl, C₁-C₄-haloalkyl,C(═O)NHR⁶, and NR⁴R⁵.

In another embodiment, X is O or NH, Y is O or NH, R³ is hydrogen and R¹is a pyrimidine ring which is substituted by one substituent R² selectedfrom the group consisting of C₁-C₄-alkyl and C(═O)NHR⁶.

In another embodiment, X is O or NH, Y is O or NH, R³ is hydrogen and R¹is a pyrimidine ring which is substituted by one substituent R² selectedfrom the group consisting of C₁-C₂-alkyl and C(═O)NHC₁-C₂-alkyl.

In another embodiment, X is O, Y is NH, R³ is hydrogen and R¹ is apyrimidine ring which is substituted by one substituent R² selected fromthe group consisting of C₁-C₂-alkyl and C(═O)NHC₁-C₂-alkyl.

In another embodiment, X is O or NH, Y is O or NH, R³ is hydrogen and R¹is a pyrimidine ring which is substituted by 0, 1, or 2 substituents R²independently selected from the group consisting of C₁-C₄-alkyl,C₁-C₄-haloalkyl, C(═O)NHR²′, and NR⁴R⁵; Ring B is selected from thegroup consisting of:

W is N or CR³; and Z is O or S; wherein R³ is H or C₁-C₄ alkyl.

In another embodiment, X is O or NH, Y is O or NH, R³ is hydrogen and R¹is a pyrimidine ring which is substituted by 0, 1, or 2 substituents R²independently selected from the group consisting of C₁-C₄-alkyl,C₁-C₄-haloalkyl, C(═O)NHR⁶, and NR⁴R⁵; Ring B is

W is N or CR³; and Z is O or S; wherein R³ is H or C₁-C₄ alkyl.

In another embodiment, X is O or NH, Y is O or NH, R³ is hydrogen and R¹is a pyrimidine ring which is substituted by 0, 1, or 2 substituents R²independently selected from the group consisting of C₁-C₄-alkyl,C₁-C₄-haloalkyl, C(═O)NHR²′, and NR⁴R⁵; Ring B is

W is N; and Z is O or S.

In another embodiment, X is O or NH, Y is O or NH, R³ is hydrogen and R¹is a pyrimidine ring which is substituted by 0, 1, or 2 substituents R²independently selected from the group consisting of C₁-C₄-alkyl andC(═O)NHR⁶; Ring B is

W is N; and Z is O or S.

In another embodiment, X is O or NH, Y is O or NH, R³ is hydrogen and R¹is a pyrimidine ring which is substituted by 0, 1, or 2 substituents R²independently selected from the group consisting of C₁-C₂-alkyl andC(═O)NH C₁-C₂-alkyl; Ring B is

W is N; and Z is O or S.

In one embodiment of compounds having formula (III-E-1) or (III-E-2),

In another embodiment,

In another embodiment, the invention features a compound selected fromthe group of compounds listed in Table 1.

In another embodiment, the invention features a compound selected fromthe group of compounds listed in Table 2.

Compositions, Formulations, and Administration of Compounds

In another embodiment, the invention provides a pharmaceuticalcomposition comprising a compound of any of the formulae describedherein and a pharmaceutically acceptable excipient. In a furtherembodiment, the invention provides a pharmaceutical compositioncomprising a compound of Table 1. In a further embodiment, thecomposition additionally comprises an additional therapeutic agent.

According to another embodiment, the invention provides a compositioncomprising a compound of this invention or a pharmaceutically acceptablederivative thereof and a pharmaceutically acceptable carrier, adjuvant,or vehicle. In one embodiment, the amount of compound in a compositionof this invention is such that is effective to measurably inhibit aDNA-PK in a biological sample or in a patient. In another embodiment,the amount of compound in the compositions of this invention is suchthat is effective to measurably inhibit DNA-PK. In one embodiment, thecomposition of this invention is formulated for administration to apatient in need of such composition. In a further embodiment, thecomposition of this invention is formulated for oral administration to apatient.

The term “patient,” as used herein, means an animal, preferably amammal, and most preferably a human.

The term “agent” is used herein to denote a chemical compound, a smallmolecule, a mixture of chemical compounds, a biological macromolecule,or an extract made from biological materials.

As used herein, “treatment” or “treating,” or “palliating” or“ameliorating” are used interchangeably. These terms refer to anapproach for obtaining beneficial or desired results including but notlimited to a therapeutic benefit and/or a prophylactic benefit. Bytherapeutic benefit is meant any therapeutically relevant improvement inor effect on one or more diseases, conditions, or symptoms undertreatment. For prophylactic benefit, the compositions may beadministered to a subject at risk of developing a particular disease,condition, or symptom, or to a subject reporting one or more of thephysiological symptoms of a disease, even though the disease, condition,or symptom may not have yet been manifested. These terms also mean thetreatment of a disease in a mammal, e.g., in a human, including (a)inhibiting the disease, i.e., arresting or preventing its development;(b) relieving the disease, i.e., causing regression of the diseasestate; or (c) curing the disease.

The term “effective amount” or “therapeutically effective amount” refersto the amount of an agent that is sufficient to effect beneficial ordesired results. The therapeutically effective amount may vary dependingupon one or more of: the subject and disease condition being treated,the weight and age of the subject, the severity of the diseasecondition, the manner of administration and the like, which can readilybe determined by one of ordinary skill in the art. The term also appliesto a dose that will provide an image for detection by any one of theimaging methods described herein. The specific dose may vary dependingon one or more of: the particular agent chosen, the dosing regimen to befollowed, whether it is administered in combination with othercompounds, timing of administration, the tissue to be imaged, and thephysical delivery system in which it is carried.

As used herein, “administer” refers to contacting, injecting,dispensing, delivering, or applying a DNA-PK inhibitor to a subject, agenomic editing system and/or a DNA-PK inhibitor to a cell or a subject.In some embodiments, the administration is contacting a genomic editingsystem and/or a DNA-PK inhibitor with a cell(s). In some embodiments,the administration is delivering a genomic editing system and/or aDNA-PK inhibitor to a cell(s). In some embodiments, the administrationis applying a genomic editing system and/or a DNA-PK inhibitor to acell(s). In some embodiments, the administration is injecting a genomicediting system and/or a DNA-PK inhibitor to a cell(s). Administering canoccur in vivo, ex vivo, or in vitro. Administering a genomic editingsystem and a DNA-PK inhibitor to a cell(s) can be done simultaneously orsequentially.

The term “acquired” in reference to a condition or disease as usedherein means a disorder or medical condition which developspost-fetally; in contrast with a congenital disorder, which is presentat birth. A congenital disorder may be antecedent to an acquireddisorder.

The terms “congenital” or “inherited” condition or disease is a geneticdisorder found in the genome of a subject that is present in a subjectat birth. The “genome” as used herein includes all of the geneticmaterial in the nucleus and the cytoplasm, and further includes themitochondrial genome and ribosomal genome. The congenital or inheritedmay be expressed at any time during the subject's life, for example atbirth or at adulthood.

The term“genetic disorder” or “genetic disease” includes inherited oracquired mutations in the genome of a subject that causes or may causedisease.

The terms “polymorphisms” or “genetic variations” means different formsof a gene at a genetic locus.

It will also be appreciated that certain of the compounds of the presentinvention can exist in free form for treatment, or where appropriate, asa pharmaceutically acceptable derivative thereof. According to thepresent invention, a pharmaceutically acceptable derivative includes,but is not limited to, pharmaceutically acceptable prodrugs, salts,esters, salts of such esters, or any other adduct or derivative whichupon administration to a patient in need is capable of providing,directly or indirectly, a compound as otherwise described herein, or ametabolite or residue thereof. As used herein, the term “inhibitoryactive metabolite or residue thereof” means that a metabolite or residuethereof is also an inhibitor of DNA-PK.

As used herein, the term “pharmaceutically acceptable salt” refers tothose salts which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and lower animalswithout undue toxicity, irritation, allergic response and the like.

Pharmaceutically acceptable salts are well known in the art. Forexample, S. M. Berge et al., describe pharmaceutically acceptable saltsin detail in J. Pharmaceutical Sciences, 66:1-19, 1977, which isincorporated herein by reference. Pharmaceutically acceptable salts ofthe compounds of this invention include those derived from suitableinorganic and organic acids and bases. Examples of pharmaceuticallyacceptable, nontoxic acid addition salts are salts of an amino groupformed with inorganic acids such as hydrochloric acid, hydrobromic acid,phosphoric acid, sulfuric acid and perchloric acid or with organic acidssuch as acetic acid, oxalic acid, maleic acid, tartaric acid, citricacid, succinic acid or malonic acid or by using other methods used inthe art such as ion exchange. Other pharmaceutically acceptable saltsinclude adipate, alginate, ascorbate, aspartate, benzenesulfonate,benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate,citrate, cyclopentanepropionate, digluconate, dodecylsulfate,ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate,gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide,2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, stearate, succinate, sulfate, tartrate,thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and thelike. Salts derived from appropriate bases include alkali metal,alkaline earth metal, ammonium and N⁺(C₁₋₄alkyl)₄ salts. This inventionalso envisions the quaternization of any basic nitrogen-containinggroups of the compounds disclosed herein. Water or oil-soluble ordispersable products may be obtained by such quaternization.Representative alkali or alkaline earth metal salts include sodium,lithium, potassium, calcium, magnesium, and the like. Furtherpharmaceutically acceptable salts include, when appropriate, nontoxicammonium, quaternary ammonium, and amine cations formed usingcounterions such as halide, hydroxide, carboxylate, sulfate, phosphate,nitrate, C₁₋₈ sulfonate and aryl sulfonate.

As described above, the pharmaceutically acceptable compositions of thepresent invention additionally comprise a pharmaceutically acceptablecarrier, adjuvant, or vehicle, which, as used herein, includes any andall solvents, diluents, or other liquid vehicle, dispersion orsuspension aids, surface active agents, isotonic agents, thickening oremulsifying agents, preservatives, solid binders, lubricants and thelike, as suited to the particular dosage form desired. In Remington: TheScience and Practice of Pharmacy, 21st edition, 2005, ed. D. B. Troy,Lippincott Williams & Wilkins, Philadelphia, and Encyclopedia ofPharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan,1988-1999, Marcel Dekker, New York, the contents of each of which isincorporated by reference herein, are disclosed various carriers used informulating pharmaceutically acceptable compositions and knowntechniques for the preparation thereof. Except insofar as anyconventional carrier medium is incompatible with the compounds of theinvention, such as by producing any undesirable biological effect orotherwise interacting in a deleterious manner with any othercomponent(s) of the pharmaceutically acceptable composition, its use iscontemplated to be within the scope of this invention.

Some examples of materials which can serve as pharmaceuticallyacceptable carriers include, but are not limited to, ion exchangers,alumina, aluminum stearate, lecithin, serum proteins, such as humanserum albumin, buffer substances such as phosphates, glycine, sorbicacid, or potassium sorbate, partial glyceride mixtures of saturatedvegetable fatty acids, water, salts or electrolytes, such as protaminesulfate, disodium hydrogen phosphate, potassium hydrogen phosphate,sodium chloride, zinc salts, colloidal silica, magnesium trisilicate,polyvinyl pyrrolidone, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, wool fat, sugars such aslactose, glucose and sucrose; starches such as corn starch and potatostarch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethyl cellulose and cellulose acetate; powdered tragacanth;malt; gelatin; talc; excipients such as cocoa butter and suppositorywaxes; oils such as peanut oil, cottonseed oil; safflower oil; sesameoil; olive oil; corn oil and soybean oil; glycols; such a propyleneglycol or polyethylene glycol; esters such as ethyl oleate and ethyllaurate; agar; buffering agents such as magnesium hydroxide and aluminumhydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer'ssolution; ethyl alcohol, and phosphate buffer solutions, as well asother non-toxic compatible lubricants such as sodium lauryl sulfate andmagnesium stearate, as well as coloring agents, releasing agents,coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the composition,according to the judgment of the formulator.

The compositions of the present invention may be administered orally,parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally or via an implanted reservoir. The term “parenteral”as used herein includes subcutaneous, intravenous, intramuscular,intra-articular, intra-synovial, intrasternal, intrathecal, intraocular,intrahepatic, intralesional, epidural, intraspinal, and intracranialinjection or infusion techniques. Preferably, the compositions areadministered orally, intraperitoneally or intravenously. Sterileinjectable forms of the compositions of this invention may be aqueous oroleaginous suspension. These suspensions may be formulated according totechniques known in the art using suitable dispersing or wetting agentsand suspending agents. The sterile injectable preparation may also be asterile injectable solution or suspension in a non-toxic parenterallyacceptable diluent or solvent, for example as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium.

For this purpose, any bland fixed oil may be employed includingsynthetic mono- or diglycerides. Fatty acids, such as oleic acid and itsglyceride derivatives are useful in the preparation of injectables, asare natural pharmaceutically-acceptable oils, such as olive oil orcastor oil, especially in their polyoxyethylated versions. These oilsolutions or suspensions may also contain a long-chain alcohol diluentor dispersant, such as carboxymethyl cellulose or similar dispersingagents that are commonly used in the formulation of pharmaceuticallyacceptable dosage forms including emulsions and suspensions. Othercommonly used surfactants, such as Tweens, Spans and other emulsifyingagents or bioavailability enhancers which are commonly used in themanufacture of pharmaceutically acceptable solid, liquid, or otherdosage forms may also be used for the purposes of formulation.

The pharmaceutically acceptable compositions of this invention may beorally administered in any orally acceptable dosage form including, butnot limited to, capsules, tablets, aqueous suspensions or solutions. Inthe case of tablets for oral use, carriers commonly used include lactoseand corn starch. Lubricating agents, such as magnesium stearate, arealso typically added. For oral administration in a capsule form, usefuldiluents include lactose and dried cornstarch. When aqueous suspensionsare required for oral use, the active ingredient is combined withemulsifying and suspending agents. If desired, certain sweetening,flavoring or coloring agents may also be added.

Alternatively, the pharmaceutically acceptable compositions of thisinvention may be administered in the form of suppositories for rectaladministration. These can be prepared by mixing the agent with asuitable non-irritating excipient that is solid at room temperature butliquid at rectal temperature and therefore will melt in the rectum torelease the drug. Such materials include cocoa butter, beeswax andpolyethylene glycols.

The pharmaceutically acceptable compositions of this invention may alsobe administered topically, especially when the target of treatmentincludes areas or organs readily accessible by topical application,including diseases of the eye, the skin, or the lower intestinal tract.Suitable topical formulations are readily prepared for each of theseareas or organs.

Topical application for the lower intestinal tract can be effected in arectal suppository formulation (see above) or in a suitable enemaformulation. Topically-transdermal patches may also be used.

For topical applications, the pharmaceutically acceptable compositionsmay be formulated in a suitable ointment containing the active componentsuspended or dissolved in one or more carriers. Carriers for topicaladministration of the compounds of this invention include, but are notlimited to, mineral oil, liquid petrolatum, white petrolatum, propyleneglycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax andwater. Alternatively, the pharmaceutically acceptable compositions canbe formulated in a suitable lotion or cream containing the activecomponents suspended or dissolved in one or more pharmaceuticallyacceptable carriers. Suitable carriers include, but are not limited to,mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax,cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

For ophthalmic use, the pharmaceutically acceptable compositions may beformulated, e.g., as micronized suspensions in isotonic, pH adjustedsterile saline or other aqueous solution, or, preferably, as solutionsin isotonic, pH adjusted sterile saline or other aqueous solution,either with or without a preservative such as benzylalkonium chloride.Alternatively, for ophthalmic uses, the pharmaceutically acceptablecompositions may be formulated in an ointment such as petrolatum. Thepharmaceutically acceptable compositions of this invention may also beadministered by nasal aerosol or inhalation. Such compositions areprepared according to techniques well-known in the art of pharmaceuticalformulation and may be prepared as solutions in saline, employing benzylalcohol or other suitable preservatives, absorption promoters to enhancebioavailability, fluorocarbons, and/or other conventional solubilizingor dispersing agents.

Most preferably, the pharmaceutically acceptable compositions of thisinvention are formulated for oral administration.

Liquid dosage forms for oral administration include, but are not limitedto, pharmaceutically acceptable emulsions, microemulsions, solutions,suspensions, syrups and elixirs. In addition to the active compounds,the liquid dosage forms may contain inert diluents commonly used in theart such as, for example, water or other solvents, solubilizing agentsand emulsifiers such as ethyl alcohol, isopropyl alcohol, ethylcarbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butylene glycol, dimethylformamide, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor, and sesame oils),glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fattyacid esters of sorbitan, and mixtures thereof. Besides inert diluents,the oral compositions can also include adjuvants such as wetting agents,emulsifying and suspending agents, sweetening, flavoring, and perfumingagents.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

The injectable formulations can be sterilized, for example, byfiltration through a bacterial-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

In order to prolong the effect of a compound of the present invention,it is often desirable to slow the absorption of the compound fromsubcutaneous or intramuscular injection. This may be accomplished by theuse of a liquid suspension of crystalline or amorphous material withpoor water solubility. The rate of absorption of the compound thendepends upon its rate of dissolution that, in turn, may depend uponcrystal size and crystalline form. Alternatively, dissolving orsuspending the compound in an oil vehicle accomplishes delayedabsorption of a parenterally administered compound form. Injectabledepot forms are made by forming microencapsule matrices of the compoundin biodegradable polymers such as polylactide-polyglycolide. Dependingupon the ratio of compound to polymer and the nature of the particularpolymer employed, the rate of compound release can be controlled.Examples of other biodegradable polymers include poly(orthoesters) andpoly(anhydrides). Depot injectable formulations are also prepared byentrapping the compound in liposomes or microemulsions that arecompatible with body tissues.

Compositions for rectal or vaginal administration are preferablysuppositories which can be prepared by mixing the compounds of thisinvention with suitable non-irritating excipients or carriers such ascocoa butter, polyethylene glycol or a suppository wax which are solidat ambient temperature but liquid at body temperature and therefore meltin the rectum or vaginal cavity and release the active compound.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activecompound is mixed with at least one inert, pharmaceutically acceptableexcipient or carrier such as sodium citrate or dicalcium phosphateand/or a) fillers or extenders such as starches, lactose, sucrose,glucose, mannitol, and silicic acid, b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia, c) humectants such as glycerol, d) disintegratingagents such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, e) solutionretarding agents such as paraffin, f) absorption accelerators such asquaternary ammonium compounds, g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, h) absorbents such as kaolinand bentonite clay, and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets and pills, thedosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like. The solid dosage forms of tablets, dragees, capsules, pills,and granules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally contain opacifying agents and can also be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner Examples of embedding compositions that can be usedinclude polymeric substances and waxes. Solid compositions of a similartype may also be employed as fillers in soft and hard-filled gelatincapsules using such excipients as lactose or milk sugar as well as highmolecular weight polethylene glycols and the like.

The active compounds can also be in micro-encapsulated form with one ormore excipients as noted above. The solid dosage forms of tablets,dragees, capsules, pills, and granules can be prepared with coatings andshells such as enteric coatings, release controlling coatings and othercoatings well known in the pharmaceutical formulating art. In such soliddosage forms the active compound may be admixed with at least one inertdiluent such as sucrose, lactose or starch. Such dosage forms may alsocomprise, as is normal practice, additional substances other than inertdiluents, e.g., tableting lubricants and other tableting aids such amagnesium stearate and microcrystalline cellulose. In the case ofcapsules, tablets and pills, the dosage forms may also comprisebuffering agents. They may optionally contain opacifying agents and canalso be of a composition that they release the active ingredient(s)only, or preferentially, in a certain part of the intestinal tract,optionally, in a delayed manner Examples of embedding compositions thatcan be used include polymeric substances and waxes.

Dosage forms for topical or transdermal administration of a compound ofthis invention include ointments, pastes, creams, lotions, gels,powders, solutions, sprays, inhalants or patches. The active componentis admixed under sterile conditions with a pharmaceutically acceptablecarrier and any needed preservatives or buffers as may be required.Ophthalmic formulation, eardrops, and eye drops are also contemplated asbeing within the scope of this invention. Additionally, the presentinvention contemplates the use of transdermal patches, which have theadded advantage of providing controlled delivery of a compound to thebody. Such dosage forms can be made by dissolving or dispensing thecompound in the proper medium. Absorption enhancers can also be used toincrease the flux of the compound across the skin. The rate can becontrolled by either providing a rate controlling membrane or bydispersing the compound in a polymer matrix or gel.

The compounds of the invention are preferably formulated in dosage unitform for ease of administration and uniformity of dosage. The expression“dosage unit form” as used herein refers to a physically discrete unitof agent appropriate for the patient to be treated. It will beunderstood, however, that the total daily usage of the compounds andcompositions of the present invention will be decided by the attendingphysician within the scope of sound medical judgment. The specificeffective dose level for any particular patient or organism will dependupon a variety of factors including the disorder being treated and theseverity of the disorder; the activity of the specific compoundemployed; the specific composition employed; the age, body weight,general health, sex and diet of the patient; the time of administration,route of administration, and rate of excretion of the specific compoundemployed; the duration of the treatment; drugs used in combination orcoincidental with the specific compound employed, and like factors wellknown in the medical arts.

The amount of the compounds of the present invention that may becombined with the carrier materials to produce a composition in a singledosage form will vary depending upon the host treated, the particularmode of administration. Preferably, the compositions should beformulated so that a dosage of between 0.01-100 mg/kg body weight/day ofthe inhibitor can be administered to a patient receiving thesecompositions.

Depending upon the particular proliferative condition or cancer to betreated, additional therapeutic agents, which are normally administeredto treat or prevent that condition, may also be present in thecompositions of this invention. As used herein, additional therapeuticagents which are normally administered to treat or prevent a particularproliferative condition or cancer are known as “appropriate for thedisease, or condition, being treated.” Examples of additionaltherapeutic agents are provided infra.

The amount of additional therapeutic agent present in the compositionsof this invention will be no more than the amount that would normally beadministered in a composition comprising that therapeutic agent as theonly active agent. Preferably the amount of additional therapeutic agentin the presently disclosed compositions will range from about 50% to100% of the amount normally present in a composition comprising thatagent as the only therapeutically active agent.

Uses of the Compounds and Compositions

In some embodiments, provided herein are methods for sensitizing a cellto a theraputic agent or a disease state that induces a DNA lesioncomprising the step of contacting the cell with one or more DNA-PKinhibitors disclosed herein, such as those of formula (III-E-1) or(III-E-2), or pharmaceutically acceptable salts thereof.

In some embodiments, provided herein are methods formethods ofpotentiating a therapeutic regimen for treatment of cancer comprisingthe step of administering to an individual in need thereof an effectiveamount of a DNA-PK inhibitors disclosed herein, such as those of formula(III-E-1) or (III-E-2) or pharmaceutically acceptable salts thereof. Inone aspect, the therapeutic regimen for treatment of cancer includesradiation therapy.

The DNA-PK inhibitors disclosed herein are useful in instances whereradiation therapy is indicated to enhance the therapeutic benefit ofsuch treatment. In addition, radiation therapy frequently is indicatedas an adjuvent to surgery in the treatment of cancer. The goal ofradiation therapy in the adjuvant setting is to reduce the risk ofrecurrence and enhance disease-free survival when the primary tumor hasbeen controlled. Adjuvant radiation therapy is indicated in severaldiseases including colon, rectal, lung, gastroesophageal, and breastcancers as described below.

In some embodiments, another anti-cancer chemotherapeutic agent with aDNA-PK inhibitor disclosed herein are used in a therapeutic regimen forthe treatment of cancer, with or without radiation therapy. Thecombination of a DNA-PK inhibitor disclosed herein with such otheragents can potentiate the chemotherapeutic protocol. For example, theDNA-PK inhibitor disclosed herein can be administered with etoposide orbleomycin, agents known to cause DNA strand breakage.

In some embodiments, further disclosed are methods for radiosensitizingtumor cells utilizing a compound of a DNA-PK inhibitor disclosed hereinADNA-PK inhibitor that can “radiosensitize” a cell, as used herein, isdefined as a molecule, preferably a low molecular weight molecule,administered to animals in therapeutically effective amount to increasethe sensitivity of cells to electromagnetic radiation and/or to promotethe treatment of diseases that are treatable with electromagneticradiation (e.g., X-rays). Diseases that are treatable withelectromagnetic radiation include neoplastic diseases, benign andmalignant tumors, and cancerous cells.

In some embodicments, also provided herein are methods for treatingcancer in an animal that includes administering to the animal aneffective amount of a DNA-PK disclosed herein. In some embodiments,further provided herein are methods of inhibiting cancer cell growth,including processes of cellular proliferation, invasiveness, andmetastasis in biological systems. Such methods include use of a DNA-PKinhibitor disclosed herein as an inhibitor of cancer cell growth. Insome specific embodiments, the methods are employed to inhibit or reducecancer cell growth, invasiveness, metastasis, or tumor incidence inliving animals, such as mammals. The DNA-PK inhibitors disclosed hereincan be used, either alone or in combination with the use of IR or one ormore chemotherapeutic agents, in treating cancer or inhibiting cancercell growth. In some embodiments, such methods are adaptable for use inassay systems, e.g., assaying cancer cell growth and properties thereof,as well as identifying compounds that affect cancer cell growth.

Tumors or neoplasms include growths of tissue cells in which themultiplication of the cells is uncontrolled and progressive. Some suchgrowths are benign, but others are termed “malignant” and can lead todeath of the organism. Malignant neoplasms or “cancers” aredistinguished from benign growths in that, in addition to exhibitingaggressive cellular proliferation, they can invade surrounding tissuesand metastasize. Moreover, malignant neoplasms are characterized in thatthey show a greater loss of differentiation (greater“dedifferentiation”) and their organization relative to one another andtheir surrounding tissues. This property is also called “anaplasia.”

Neoplasms treatable by the present invention also include solid tumors,i.e., carcinomas and sarcomas. Carcinomas include those malignantneoplasms derived from epithelial cells which infiltrate (invade) thesurrounding tissues and give rise to metastases. Adenocarcinomas arecarcinomas derived from glandular tissue, or from tissues which formrecognizable glandular structures. Another broad category of cancersincludes sarcomas, which are tumors whose cells are embedded in afibrillar or homogeneous substance like embryonic connective tissue. TheDNA-PK hibitors disclosed herein can also enables treatment of cancersof the myeloid or lymphoid systems, including leukemias, lymphomas, andother cancers that typically do not present as a tumor mass, but aredistributed in the vascular or lymphoreticular systems.

DNA-PK activity can be associated with various forms of cancer in, forexample, adult and pediatric oncology, growth of solidtumors/malignancies, myxoid and round cell carcinoma, locally advancedtumors, metastatic cancer, human soft tissue sarcomas, including Ewing'ssarcoma, cancer metastases, including lymphatic metastases, squamouscell carcinoma, particularly of the head and neck, esophageal squamouscell carcinoma, oral carcinoma, blood cell malignancies, includingmultiple myeloma, leukemias, including acute lymphocytic leukemia, acutenonlymphocytic leukemia, chronic lymphocytic leukemia, chronicmyelocytic leukemia, and hairy cell leukemia, effusion lymphomas (bodycavity based lymphomas), thymic lymphoma lung cancer, including smallcell lung carcinoma, cutaneous T cell lymphoma, Hodgkin's lymphoma,non-Hodgkin's lymphoma, cancer of the adrenal cortex, ACTH-producingtumors, nonsmall cell cancers, breast cancer, including small cellcarcinoma and ductal carcinoma, gastrointestinal cancers, includingstomach cancer, colon cancer, colorectal cancer, polyps associated withcolorectal neoplasia, pancreatic cancer, liver cancer, urologicalcancers, including bladder cancer, including primary superficial bladdertumors, invasive transitional cell carcinoma of the bladder, andmuscle-invasive bladder cancer, prostate cancer, malignancies of thefemale genital tract, including ovarian carcinoma, primary peritonealepithelial neoplasms, cervical carcinoma, uterine endometrial cancers,vaginal cancer, cancer of the vulva, uterine cancer and solid tumors inthe ovarian follicle, malignancies of the male genital tract, includingtesticular cancer and penile cancer, kidney cancer, including renal cellcarcinoma, brain cancer, including intrinsic brain tumors,neuroblastoma, astrocytic brain tumors, gliomas, metastatic tumor cellinvasion in the central nervous system, bone cancers, including osteomasand osteosarcomas, skin cancers, including malignant melanoma, tumorprogression of human skin keratinocytes, squamous cell cancer, thyroidcancer, retinoblastoma, neuroblastoma, peritoneal effusion, malignantpleural effusion, mesothelioma, Wilms's tumors, gall bladder cancer,trophoblastic neoplasms, hemangiopericytoma, and Kaposi's sarcoma.Methods to potentiate treatment of these and other forms of cancer arealso disclosed herein.

Also provided herein are methods for inhibiting DNA-PK activity in abiological sample that includes contacting the biological sample with acompound or composition of the invention. The term “biological sample,”as used herein, means a sample outside a living organism and includes,without limitation, cell cultures or extracts thereof; biopsied materialobtained from a mammal or extracts thereof; and blood, saliva, urine,feces, semen, tears, or other body fluids or extracts thereof.Inhibition of kinase activity, particularly DNA-PK activity, in abiological sample is useful for a variety of purposes known to one ofskill in the art. Examples of such purposes include, but are not limitedto, biological specimen storage and biological assays. In oneembodiment, the method of inhibiting DNA-PK activity in a biologicalsample is limited to non-therapeutic methods.

In some embodiments, this disclosure provides methods, compositions andkits for editing a target genome, e.g., by correcting a mutation. Suchmethods, compositions and kits can increase genome editing efficiency bythe use of a DNA-PK inhibitor.

A genomic editing system can stimulate or induce a DNA break(s), such asDSB(s) at the desired locus in the genome (or target genomic region).The creation of DNA cleavage prompts cellular enzymes to repair the siteof break through either the error prone NHEJ pathway or through theerror-free HDR pathway. In NHEJ, the DNA lesion is repaired by fusingthe two ends of the DNA break in a series of enzymatic processesinvolving Ku70/80 heterodimer and DNA dependent protein kinase (DNA-PK)enzymes. The repair mechanism involves tethering and alignment of twoDNA ends, resection, elongation and ligation (Rouet et al.; Dexheimer T.DNA repair pathways and mechanisms. In: Mathews L, Cabarcas S, Hurt E,editors. DNA repair of cancer stem cells. Dordrecht: Springer; 2013. p.19-32.) resulting in the formation of small insertion or deletionmutations (indels) at the break site. Indels introduced into the codingsequence of a gene can cause either premature stop codon or frame-shiftmutations that lead to the production of nonfunctional, truncatedproteins. The mechanism of HDR pathway is less understood and involves adifferent set of repair proteins such as Rad51 that stimulate strandinvasion by a donor repair template for base insertion or genereplacement. Hence, HDR allows introduction of exogenous DNA template toobtain a desired outcome of DNA editing within a genome and can be apowerful strategy for translational disease modeling and therapeuticgenome editing to restore gene function.

Of the two DNA repair pathways, NHEJ occurs at a much higher frequencyand reports of more than 70% efficiency can be achieved even in neurons(Swiech et al., “In vivo interrogation of gene function in the mammalianbrain using CRISPR-Cas9,” Nat Biotechnol. 2015 January;33(1):102-62014). The HDR gene correction however, occurs at very lowfrequency and during S and G2 phase when DNA replication is completedand sister chromatids are available to serve as repair templates (Heyeret al., Regulation of homologous recombination in eukaryotes. AnnualReview of Genetics 44:113-139, 2010). Since NHEJ occurs throughout thecell cycle, in competition and is favored over HDR during the S and G2phase, targeted insertion through the HDR pathway remains a challengeand a focus of continued studies.

DNA protein-kinase (DNA-PK) plays a role in various DNA repairprocesses. DNA-PK participates in DNA double-stranded break repairthrough activation of the nonhomologous end-joining (NHEJ) pathway. NHEJis thought to proceed through three steps: recognition of the DSBs, DNAprocessing to remove non-ligatable ends or other forms of damage at thetermini, and finally ligation of the DNA ends. Recognition of the DSB iscarried out by binding of the Ku heterodimer to the ragged DNA endsfollowed by recruitment of two molecules of DNA-dependent protein kinasecatalytic subunit (DNA-PKcs) to adjacent sides of the DSB; this servesto protect the broken termini until additional processing enzymes arerecruited. Recent data supports the hypothesis that DNA-PKcsphosphorylates the processing enzyme, Artemis, as well as itself toprepare the DNA ends for additional processing. In some cases DNApolymerase may be required to synthesize new ends prior to the ligationstep. The auto-phosphorylation of DNA-PKcs is believed to induce aconformational change that opens the central DNA binding cavity,releases DNA-PKcs from DNA, and facilitates the ultimate re-ligation ofthe DNA ends.

In some embodiments, this disclosure provides methods, compositions, andkits to enhance gene editing, in particular increasing the efficiency ofrepair of DNA break(s) via a HDR pathway, or the efficiency ofinhibiting or suppressing repair of DNA break(s) via a NHEJ pathway, ingenome editing systems, including CRISPR-based HDR repair in cells.While not being bound by a particular theory, it is believed that agenome editing system administered to a cell(s) interacts with a nucleicacid(s) of the target gene, resulting in or causing a DNA break; suchDNA break is repaired by several repair pathways, e.g., HDR, and aDNA-PK inhibitor administered to a cell(s) inhibits, blocks, orsuppresses a NHEJ repair pathway, and the frequency or efficiency of HDRDNA repair pathway can be increased or promoted.

The interaction between a genome editing system with a nucleic acid(s)of the target gene can be hybridization of at least part of the genomeediting system with the nuclecic acid(s) of the target gene, or anyother recognition of the nuclecic acid(s) of the target gene by thegenone editing system. In some embodiments, such interaction is aprotein-DNA interactions or hybridization between base pairs.

In some embodiments, this disclosure provides methods of editing one ormore target genomic regions in a cell(s) by administering to the cell(s)a genome editing system and a DNA-PK inhibitor. The editing can occursimultaneously or sequentially. Editing of the one or more targetgenomic regions includes any kind of genetic manipulations orengineering of a cell's genome. In some embodiments, the editing of theone or more target genomic regions can include insertions, deletions, orreplacements of genomic regions in a cell(s). Genomic regions comprisethe genetic material in a cell(s), such as DNA, RNA, polynucleotides,and oligonucleotides. Genomic regions in a cell(s) also comprise thegenomes of the mitochondria or chloroplasts contained in a cell(s).

In some embodiments, the insertions, deletions or replacements can beeither in a coding or a non-coding genomic region, in intronic or exonicregions, or any combinations thereof including overlapping ornon-overlapping segments thereof. As used herein, a “non-coding region”refers to genomic regions that do not encode an amino acid sequence. Forexample, non-coding regions include introns. Coding regions refer togenomic regions that code for an amino acid sequence. For example,coding regions include exons.

In some embodiments, the editing of one or more target genomic regionscan occur in any one or more target regions in a genome of a cell(s). Insome embodiments, the editing of one or more target genomic regions canoccur, for example, in an exon, an intron, a transcription start site,in a promoter region, an enhancer region, a silencer region, aninsulator region, an antirepressor, a post translational regulatoryelement, a polyadenylation signal (e.g. minimal poly A), a conservedregion, a transcription factor binding site, or any combinationsthereof.

In some embodiments, administration to a cell(s) with a DNA-PK inhibitorand a genomic editing system results in increased targeted genomeediting efficiency as compared to conditions in which a DNA-PK inhibitorand a genomic editing system is not administered to a cell(s). In someembodiments, the increased editing efficiency is about 1-fold, 2-fold,3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold,40-fold, 50-fold, or 100-fold, in comparison to a condition in which aDNA-PK inhibitor and a genome editing system is not administered to acell(s), or compared to a condition in which only a genome editingsystem and not a DNA-PK inhibitor is administered to a cell(s). Theefficiency of genomic editing can be measured by any method known in theart, for example, by any method that ascertains the frequency oftargeted polynucleotide integration or by measuring the frequency oftargeted mutagenesis. Targeted polynucleotide integrations can alsoresult in alteration or replacement of a sequence in a genome,chromosome or a region of interest in cellular chromatin. Targetedpolynucleotide integrations can result in targeted mutations including,but not limited to, point mutations (i.e., conversion of a single basepair to a different base pair), substitutions (i.e., conversion of aplurality of base pairs to a different sequence of identical length),insertions or one or more base pairs, deletions of one or more basepairs and any combination of the aforementioned sequence alterations.

In some embodiments, the methods of editing one or more target genomicregions in a cell(s) involve administering to the cell(s) a genomeediting system and a DNA-PK inhibitor. In some embodiments, the cell(s)is synchronized at the S or the G2 cell cycle phase. Synchronization ofthe cell(s) at the S or G2 cell cycle phase can be achieved by anymethod known in the art. As a non-limiting example, agents that can beused to synchronize a cell(s) at the S or G2 cell cycle phase includeaphidicolin, dyroxyurea, lovastatin, mimosine, nocodazole, thymidine, orany combinations thereof. (See, Lin et al.“Enhanced homology-directedhuman genome engineering by controlled timing of CRISPR/Cas9 delivery,”Elife. 2014 Dec. 15; 3). In some embodiments, the agents for cellsynchronization can be administered at any time during the gene-editingprocess. In some embodiments, a cell(s) can be synchronized at the S orthe G2 phase of the cell cycle before, during, or after administering toa cell(s) a genome editing system and/or a DNA-PK inhibitor.

In some embodiments, the methods of editing one or more target genomicregions in a cell(s) by administering to the cell(s) a genome editingsystem and a DNA-PK inhibitor results in increased cell survival incomparison to conditions in which a genome editing system and a DNA-PKinhibitor were not administered to a cell(s), or in comparison toconditions in which only a gene editing system is contacted oradministered into a cell(s) and not a DNA-PK inhibitor.

In some embodimetns, provided herein are methods of repairing a DNAbreak in one or more target genomic regions via an HDR pathway. Theadministering to a cell(s) a genome editing system and a DNA-PKinhibitor results in a DNA break of a targeted region of the genome, andthe DNA break is subsequently repaired, at least in part, by a HDRpathway. These methods result in increased amounts of HDR-mediatedrepair (e.g. HDR pathway) in the one or more target genomic regionsresulting in greater efficiency of HDR-mediated repair as compared toconditions in which a DNA-PK inhibitor and a genomic editing system isnot administered to a cell(s). In some embodiments, the efficiency ofHDR pathway mediated repair of the DNA break is about 1-fold, 2-fold,3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold,40-fold, 50-fold, or 100-fold, in comparison to a condition in which aDNA-PK inhibitor and a genome editing system is not administered to acell(s), or compared to a condition in which only a genome editingsystem and not a DNA-PK inhibitor is administered to a cell(s). Theefficiency of HDR pathway mediated repair can be measured by any methodknown in the art, for example, by ascertaining the frequency of targetedpolynucleotide integration or by measuring the frequency of targetedmutagenesis.

In some embodiments, the methods herein provide for repairing the DNAbreak by increasing the efficiency of the HDR pathway.

The HDR pathway can be “canonical” or “alternative.” “HDR” (homologydirected repair) refers to a specialized form of DNA repair that takesplace, for example, during repair of double-strand breaks or a DNA nickin a cell(s). HDR of double stranded breaks is generally based onnucleotide sequence homology, uses a “donor” molecule to template repairof a “target” molecule (e.g., the one that experienced the double-strandbreak), and can lead to the transfer of genetic information from thedonor to the target. Canonical HDR of double stranded breaks isgenerally based on BRCA2 and RAD51 and typically employs a dsDNA donormolecule. Non-canonical, or “alternative,” HDR is an HDR mechanism thatis suppressed by BRCA2, RAD51, and/or functionally-related genes.Alternative HDR may use a ssDNA or nicked dsDNA donor molecule. See, forexample, WO 2014172458.

In some embodiments, the methods of repairing a DNA break in one or moretarget genomic regions via an HDR pathway by administering to thecell(s) a genome editing system and a DNA-PK inhibitor result inincreased cell survival in comparison to conditions in which a genomeediting system and a DNA-PK inhibitor are not administered to a cell(s),or in comparison to conditions in which only a gene editing system isadministered to a cell(s) and not a DNA-PK inhibitor.

In some embodiments, provided herein are methods of inhibiting orsuppressing NHEJ-mediated repair of a DNA break in one or more targetgenomic regions in a cell(s). In some embodiments, the inhibiting orsuppressing of NHEJ-mediated repair of a DNA break is performed byinhibiting or suppressing the NHEJ pathway. The NHEJ pathway can beeither classical (“canonical”) or an alternative NHEJ pathway (alt-NHEJ,or microhomology-mediated end joining (MMEJ)). The NHEJ pathway oralt-NHEJ pathway is suppressed in a cell(s) by administering to acell(s) a genome editing system and a DNA-PK inhibitor.

The classical NHEJ repair pathway is a DNA double stranded break repairpathway in which the ends of the double stranded break are ligatedwithout extensive homology. Classical NHEJ repair uses several factors,including KU70/80 heterodimer (KU), XRCC4, Ligase IV, and DNA proteinkinases catalytic subunit (DNA-PKcs). Alt-NHEJ is another pathway forrepairing double strand breaks. Alt-NHEJ uses a 5-25 base pairmicrohomologous sequence during alignment of broken ends before joiningthe broken ends. Alt-NHEJ is largely independent of KU70/80 heterodimer(KU), XRCC4, Ligase IV, DNA protein kinases catalytic subunit(DNA-PKcs), RAD52, and ERCC1. See, Bennardo et al., “Alternative-NHEJ isa Mechanistically Distinct Pathway of Mammalian Chromosome BreakRepair,” PLOS Genetics, Jun. 27, 2008.

In some embodiments, the methods of inhibiting or suppressingNHEJ-mediated repair of a DNA break via the NHEJ pathway in one or moretarget genomic regions in a cell(s) by inhibiting or suppressing theNHEJ pathway though the administering to a cell(s) a genomic editingsystem and a DNA-PK inhibitor result in increased efficiency ofinhibiting or suppressing the NHEJ-mediated repair of the DNA break incomparison to a cell(s) that have not received a genomic editing systemand a DNA-PK inhibitor, or in comparison to a condition in which acell(s) receives a genomic editing system and not a DNA-PK inhibitor. Insome embodiments, the increased efficiency of inhibiting or suppressingrepair of a DNA break via the NHEJ pathway by contacting a cell(s) witha DNA-PK inhibitor and a genome editing system is about 1-fold, 2-fold,3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold,40-fold, 50-fold, or 100-fold, in comparison to a condition in which aDNA-PK inhibitor and a genome editing system is not administered to acell(s), or compared to a condition in which only a genome editingsystem and not a DNA-PK inhibitor is administered to a cell(s). Theefficiency inhibiting or suppressing repair of a DNA break via the NHEJpathway can be measured by any method known in the art, for example, byascertaining the frequency of targeted polynucleotide integration or bymeasuring the frequency of targeted mutagenesis.

In some embodiments, the methods of inhibiting or suppressingNHEJ-mediated repair of a DNA break in one or more target genomicregions in a cell(s) by inhibiting or suppressing the NHEJ pathwaythough the administering to a cell(s) a genomic editing system and aDNA-PK inhibitor result in increased cell survival in comparison toconditions in which a genome editing system and a DNA-PK inhibitor werenot contacted or administered to a cell(s), or in comparison toconditions in which only a gene editing system is contacted oradministered into a cell(s) and not a DNA-PK inhibitor.

The DNA break can be a double stranded break (DSB) or two singlestranded breaks (e.g. two DNA nicks). The DSB can be blunt ended or haveeither a 5′ or 3′ overhang, if the strands are each cleaved too farapart, the overhangs will continue to anneal to each other and exist astwo nicks, not one DSB.

In some embodiments, provided herein are methods of modifying expressionof one or more genes (a target gene(s)), and/or corresponding ordownstream proteins, by administering to a cell(s) a genome editingsystem and a DNA-PK inhibitor. In some embodiments, the genome editingsystem can create, for example, insertions, deletions, replacements,modiication or disruption in a target genomic region(s) of a targetgene(s) of the cell(s), resulting in modified expression of the targetgene(s). In some embodiments, the insertion, deletions, replacement,modification or disruption can result in targeted expression of aspecific protein, or group of proteins, or of downstream proteins. Insome embodiments, the genome editing system can create insertions,deletions or replacements in non-coding regions or coding regions. Insome embodiments, the genome editing system can create insertions,deletions, replacements, modification or disruption in a promoterregion, enhancer region, and/or any other gene regulatory element,including an exon, an intron, a transcription start site, a silencerregion, an insulator region, an antirepressor, a post translationalregulatory element, a polyadenylation signal (e.g. minimal poly A), aconserved region, a transcription factor binding site, or anycombinations thereof. In some embodiments, the genome editing system cancreate the insertions, deletions, replacements, modification ordisruption in more than one target region, simultaneously orsequentially. In some embodiments, administering to a cell(s) with agenome editing system and a DNA-PK inhibitor can allow for targetedmodified gene expression in the cell(s). Such targeted modified geneexpression can lead to expression of specific proteins and downstreamproteins thereof.

In some embodiments, the expression of a downstream gene and/or proteinis increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1,1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold,or 10-fold in comparison to a condition in which a DNA-PK inhibitor anda genome editing system is not administered to a cell(s), or compared toa condition in which only a genome editing system and not a DNA-PKinhibitor is administered to a cell(s).

In some embodiments, the gene expression of a downstream gene and/orprotein is decreased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 95%, 96%, 97%, 98%, or 99% in comparison to a condition in which aDNA-PK inhibitor and a genome editing system is not administered to acell(s), or compared to a condition in which only a genome editingsystem and not a DNA-PK inhibitor is administered to a cell(s).

The cell of the methods herein can be any cell. In some embodiments, thecell is a vertebrate cell. In some embodiments, the vertebrate cell is amammalian cell. In some embodiment, the vertebrate cell is a human cell.

The cell can be any kind of cell at any developmental stage. In someembodiments, the cell can be a differentiated cell, a totipotent stemcell, a pluripotent stem cell, an embryonic stem cell, an embryonic germcell, an adult stem cell, a precursor cell, an induced pluripotent stemcell, or any combinations thereof. A differentiated cell is aspecialized cell that performs a specific function in a tissue. Atotipotent stem cell is an undifferentiated cell from an embryo, fetusor adult that can divide for extended periods and has the capability ofdifferentiating into any cell type of any of the three germ layers of anorganism. A pluripotent stem cell is an undifferentiated cell from anembryo, fetus or adult that can divide for extended periods and has thecapability of differentiating into any cell type of an organism exceptextra-embryonic tissue or the placenta. An embryonic stem cell is anundifferentiated stem cell that is found in the inner cell mass of anembryo and has the capability to differentiate into any type of cell ofany of the three germ layers. An embryonic germ cell is an embryoniccell that can give rise to reproductive cells, such as sperm cells oregg cells. An adult stem cell is an undifferentiated cell that is foundin differentiated tissue, is capable of self-renewal and candifferentiate into any of the cells of the tissue in which it resides. Aprecursor or progenitor cell is a partially differentiated cell whichtypically can only differentiate into one kind of cell (e.g. a unipotentcell). An induced pluripotent stem cell is a kind of pluripotent stemcell that is generated from an adult differentiated or partiallydifferentiated cell. See, for example, WO/2010/017562.

As used herein, the singular form “a”, “an” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a cell” includes a plurality of cells, including mixturesthereof. For example “one or more cells” and “a cell(s)” areinterchangeably used herein. Similarly, “one or more target genomicregions” and “a target genomic region(s)” are interchangeably usedherein.

The terms, “approximately” and “about” are used interchangeably herein.The term “approximately” or “about,” as applied to one or more values ofinterest, refers to a value that is similar to a stated reference value.In certain embodiments, the term “approximately” or “about” refers to arange of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%,13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less ineither direction (greater than or less than) of the stated referencevalue unless otherwise stated or otherwise evident from the context(except where such number would exceed 100% of a possible value).

The terms “polynucleotide”, “nucleotide”, “nucleotide sequence”,“nucleic acid” and “oligonucleotide” are used interchangeably. Theyrefer to a polymeric form of nucleotides of any length, eitherdeoxyribonucleotides (DNA) or ribonucleotides (RNA), or analogs thereof.Polynucleotides may have any three dimensional structure, and mayperform any function, known or unknown. The following are non-limitingexamples of polynucleotides: coding or non-coding regions of a gene orgene fragment, loci (locus) defined from linkage analysis, exons,introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, shortinterfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA),ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides,plasmids, vectors, isolated DNA of any sequence, isolated RNA of anysequence, nucleic acid probes, and primers. A polynucleotide maycomprise one or more modified nucleotides, such as methylatednucleotides and nucleotide analogs. If present, modifications to thenucleotide structure may be imparted before or after assembly of thepolymer. The sequence of nucleotides may be interrupted bynon-nucleotide components. A polynucleotide may be further modifiedafter polymerization, such as by conjugation with a labeling component.The term “ssDNA” means a single stranded DNA molecule. The term “ssODN”means single stranded oligodeoxynucleotides.

The term “naturally occurring nucleotides” referred to herein includesdeoxyribonucleotides and ribonucleotides. The term “modifiednucleotides” referred to herein includes nucleotides with modified orsubstituted sugar groups and the like. The term “oligonucleotidelinkages” referred to herein includes oligonucleotides linkages such asphosphorothioate, phosphorodithioate, phosphoroselerloate,phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate,phosphoronmidate, and the like. An oligonucleotide can include a labelfor detection, if desired.

The term “synthetic RNA” refers to RNA that is engineered ornon-naturally occurring.

As used herein the term “wild type” is a term of the art understood byskilled persons and means the typical form of an organism, strain, geneor characteristic as it occurs in nature as distinguished from mutant orvariant forms.

The terms “non-naturally occurring” or “engineered” are usedinterchangeably and indicate the involvement of the hand of man. Theterms, when referring to nucleic acid molecules or polypeptides meanthat the nucleic acid molecule or the polypeptide is at leastsubstantially free from at least one other component with which they arenaturally associated in nature and as found in nature.

“Complementarity” refers to the ability of a nucleic acid to formhydrogen bond(s) with another nucleic acid by either traditionalWatson-Crick or other non-traditional types. A percent complementarityindicates the percentage of residues in a nucleic acid molecule whichcan form hydrogen bonds (e.g., Watson-Crick base pairing) with a secondnucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%,70%, 80%, 90%, and 100% complementary). “Perfectly complementary” meansthat all the contiguous residues of a nucleic acid sequence willhydrogen bond with the same number of contiguous residues in a secondnucleic acid sequence. “Substantially complementary” as used hereinrefers to a degree of complementarity that is at least 60%, 65%, 70%,75%, 80%, 85%, 90%. 95%, 97%, 98%, 99%, or 100% over a region of 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35,40, 45, 50, or more nucleotides, or refers to two nucleic acids thathybridize under stringent conditions.

As used herein, “expression” refers to the process by which apolynucleotide is transcribed from a DNA template (such as into mRNA orother RNA transcript) and/or the process by which a transcribed mRNA issubsequently translated into peptides, polypeptides, or proteins.Transcripts and encoded polypeptides may be collectively referred to as“gene product.” If the polynucleotide is derived from genomic DNA,expression may include splicing of the mRNA in a eukaryotic cell.

The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably herein to refer to polymers of amino acids of anylength. The polymer may be linear or branched, it may comprise modifiedamino acids, and it may be interrupted by non-amino acids. The termsalso encompass an amino acid polymer that has been modified; forexample, disulfide bond formation, glycosylation, lipidation,acetylation, phosphorylation, or any other manipulation, such asconjugation with a labeling component. As used herein the term “aminoacid” includes natural and/or unnatural or synthetic amino acids,including glycine and both the D or L optical isomers, and amino acidanalogs and peptidomimetics.

A “viral vector” is defined as a recombinantly produced virus or viralparticle that comprises a polynucleotide to be delivered into a hostcell, either in vivo, ex vivo or in vitro. Examples of viral vectorsinclude retroviral vectors, adenoviral vectors, adeno-associated virusvectors, adenoviral vectors, lentiviral vectors, herpes simplex viralvectors, and chimeric viral vectors and the like. In some embodimentsswhere gene transfer is mediated by a retroviral vector, a vectorconstruct refers to the polynucleotide comprising the retroviral genomeor part thereof.

Some embodiments of the disclosure relate to vector systems comprisingone or more vectors, or vectors as such. Vectors can be designed forexpression of CRISPR transcripts (e.g. nucleic acid transcripts,proteins, or enzymes) in prokaryotic or eukaryotic cells. For example,CRISPR transcripts can be expressed in bacterial cells such asEscherichia coli, insect cells (using baculovirus expression vectors),yeast cells, or mammalian cells.

The cells can be primary cells, induced pluripotent stem cells (iPSCs),embryonic stem cells (hESCs), adult stem cells, progenitor cells or celllines. “Primary cells” are cells taken directly from living tissue andplaced in vitro for growth. Primary cells have few population doublings,and have a finite lifespan for population doublings in vitro. “Stemcells,” “embryonic stem cells,” and “induced pluripotent stem cells,”are unspecialized and undifferentiated cells capable of self-renewal andhaving the potential to differentiate into cells of different types withspecialized function. “Cell lines” include cell cultures that arederived from one cell type or a set of cells of the same type which canproliferate indefinitely. Non-limiting examples of mammalian cell linescan include CD34 cells, 293 cells, HEK cells, CHO cells, BHK cells, CV-1cells, Jurkat cells, HeLa cells, or any variants thereof.

In some embodiments, a vector is capable of driving expression of one ormore sequences in mammalian cells using a mammalian expression vector.Examples of mammalian expression vectors include pCDM8 and pMT2PC. Whenused in mammalian cells, the expression vector's control functions aretypically provided by one or more regulatory elements. For example,commonly used promoters are derived from polyoma, adenovirus 2,cytomegalovirus, simian virus 40, and others disclosed herein and knownin the art. Other promoters can include, for example, EF1 promoter, orEF1 alpha promoter. For other suitable expression systems for bothprokaryotic and eukaryotic cells see, e.g., Chapters 16 and 17 ofSambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., ColdSpring Harbor Laboratory, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989.

As used herein, the terms “label” or “labeled” refers to incorporationof a detectable marker, e.g., by incorporation of a radiolabeled aminoacid or attachment to a polypeptide of biotinyl moieties that can bedetected by marked avidin (e.g., streptavidin containing a fluorescentmarker or enzymatic activity that can be detected by optical orcalorimetric methods). In certain situations, the label or marker canalso be therapeutic. Various methods of labeling polypeptides andglycoproteins are known in the art and may be used. Examples of labelsfor polypeptides include, but are not limited to, the following:radioisotopes or radionuclides (e.g., ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc,¹¹¹In, ¹²⁵I, ¹³¹I) fluorescent labels (e.g., FITC, rhodamine, lanthanidephosphors), enzymatic labels (e.g., horseradish peroxidase,p-galactosidase, luciferase, alkaline phosphatase), chemiluminescent,biotinyl groups, predetermined polypeptide epitopes recognized by asecondary reporter (e.g., leucine zipper pair sequences, binding sitesfor secondary antibodies, metal binding domains, epitope tags). In someembodiments, labels are attached by spacer arms of various lengths toreduce potential steric hindrance. The term “pharmaceutical agent ordrug” as used herein refers to a chemical compound or compositioncapable of inducing a desired therapeutic effect when properlyadministered to a patient.

As used herein, “substantially pure” means an object species is thepredominant species present (i.e., on a molar basis it is more abundantthan any other individual species in the composition). In someembodiments, a substantially purified fraction is a composition whereinthe object species comprises at least about 50 percent (on a molarbasis) of all macromolecular species present.

Generally, a substantially pure composition will comprise more thanabout 80 percent of all macromolecular species present in thecomposition. In some embodiments, a substantially pure composition willcomprise more than about 85%, 90%, 95%, and 99% of all macromolecularspecies present in the composition. In some embodiments, the objectspecies is purified to essential homogeneity (contaminant species arenot detected in the composition by conventional detection methods)wherein the composition consists essentially of a single macromolecularspecies.

Genome Editing System

Various types of genome engineering systems can be used. The terms“genome editing system,” “gene editing system,” and the like, are usedinterchangeably herein, and refer to a system or technology which editsa target gene or the function or expression thereof. A genome editingsystem comprises: at least one endonuclease component enabling cleavageof a target genomic region(s) (or target sequence(s)); and at least onegenome-targeting element which brings or targets the endonucleasecomponent to a target genomic region(s). Examples of genome-targetingelement include a DNA-binding domain (e.g., zinc finger DNA-bindingprotein or a TALE DNA-binding domain), guide RNA elements (e.g., CRISPRguide RNA), and guide DNA elements (e.g., NgAgo guide DNA). Programmablegenome-targeting and endonuclease elements enable precise genome editingby introducing DNA breaks, such as double strand breaks (DSBs) atspecific genomic loci. DSBs subsequently recruit endogenous repairmachinery for either non-homologous end-joining (NHEJ) or homologydirected repair (HDR) to the DSB site to mediate genome editing. The“endonuclease component” comprises an endonuclease or a nucleic acidcomprising a nucleotide sequence(s) encoding such endonuclease.

The term “endonuclease” refers to any wild-type, mutant, variant, orengineered enzyme capable of catalyzing the hydrolysis (cleavage) of abond between nucleic acids within a DNA or RNA molecule. Endonucleasescan recognize and cleave a DNA or RNA molecule at its target genomicregions. Examples of endonucleases include a homing endonuclease;restriction enzyme such as FokI; a chimeric Zinc-Finger nuclease (ZFN)resulting from the fusion of engineered zinc-finger domains with thecatalytic domain of a restriction enzyme such as FokI; Cas enzymes, andCpf enzymes. Chemical endonucleases in which a chemical or peptidiccleaver is conjugated either to a polymer of nucleic acids or to anotherDNA recognizing a specific target sequence, thereby targeting thecleavage activity to a specific sequence, are comprised in the term“endonuclease”. Examples of chemical enonucleases include syntheticnucleases like conjugates of orthophenanthroline, a DNA cleavingmolecule, and triplex-forming oligonucleotides (TFOs).

By “variant” it is intended a recombinant protein obtained byreplacement of at least one residue in the amino acid sequence of theparent protein with a different amino acid.

In some embodiments, endonucleases such as ZFNs, TALENs and/ormeganucleases comprise a cleavage domain and/or cleavage half-domain.The cleavage domain may be homologous or heterologous to the DNA-bindingdomain. For example, a zinc finger DNA-binding domain and a cleavagedomain from a nuclease or a meganuclease DNA-binding domain and cleavagedomain from a different nuclease can be used. Heterologous cleavagedomains can be obtained from any endonuclease or exonuclease. Exemplaryendonucleases from which a cleavage domain can be derived include, butare not limited to, restriction endonucleases and homing endonucleases.See, for example, WO2013/130824. Additional enzymes which cleave DNA areknown (e.g., S1 Nuclease; mung bean nuclease; pancreatic DNase I;micrococcal nuclease; yeast HO endonuclease; see also Linn et al. (eds.)Nucleases, Cold Spring Harbor Laboratory Press, 1993). One or more ofthese enzymes (or functional fragments thereof) can be used as a sourceof cleavage domains and cleavage half-domains.

A cleavage half-domain can be derived from any nuclease or portionthereof, as set forth above, that requires dimerization for cleavageactivity. In some embodiments, two fusion proteins are required forcleavage if the fusion proteins comprise cleavage half-domains. In someembodiments, a single protein comprising two cleavage half-domains canbe used. In some embodiments, the two cleavage half-domains can bederived from the same endonuclease (or functional fragments thereof). Insome embodiments, each cleavage half-domain can be derived from adifferent endonuclease (or functional fragments thereof). In addition,the target sites for the two fusion proteins are preferably disposed,with respect to each other, such that binding of the two fusion proteinsto their respective target sites places the cleavage half-domains in aspatial orientation to each other that allows the cleavage half-domainsto form a functional cleavage domain, e.g., by dimerizing. Thus, incertain embodiments, the near edges of the target sites are separated by5-50 nucleotides, 5-8 nucleotides or by 15-18 nucleotides. It is notedthat any integral number of nucleotides or nucleotide pairs canintervene between two target sites (e.g., from 2 to 50 nucleotide pairsor more). In some embodiments, the site of cleavage lies between thetarget sites.

Restriction endonucleases (restriction enzymes) are present in manyspecies and are capable of sequence-specific binding to DNA (at arecognition site), and cleaving DNA at or near the site of binding.Certain restriction enzymes (e.g., Type IIS) cleave DNA at sites removedfrom the recognition site and have separable binding and cleavagedomains. For example, the Type IIS enzyme Fok I catalyzesdouble-stranded cleavage of DNA. See, for example, U.S. Pat. Nos.5,356,802; 5,436,150 and 5,487,994; as well as Li et al. (1992) Proc.Natl. Acad. Sci. USA 89:4275-4279; Li et al. (1993) Proc. Natl. Acad.Sci. USA 90:2764-2768; Kim et al. (1994a) Proc. Natl. Acad. Sci. USA91:883-887; Kim et al. (1994b) J. Biol. Chem. 269:31,978-31,982.

In some embodiments, the endonuclease component comprises a fusionprotein(s) that include a cleavage domain (or cleavage half-domain) fromat least one Type IIS restriction enzyme and one or more zinc fingerbinding domains, which may or may not be engineered. An exemplary TypeIIS restriction enzyme, whose cleavage domain is separable from thebinding domain, is Fok I. This particular enzyme is active as a dimer.Bitinaite et al. (1998) Proc. Natl. Acad. Sci. USA 95: 10,570-10,575.The portion of the Fok I enzyme used in such fusion proteins isconsidered a cleavage half-domain. Thus, for targeted double-strandedcleavage and/or targeted replacement of cellular sequences using zincfinger- or TALE-Fok I fusions, two fusion proteins, each comprising aFokI cleavage half-domain, can be used to reconstitute a catalyticallyactive cleavage domain. Alternatively, a single polypeptide moleculecontaining a zinc finger binding domain and two Fok I cleavagehalf-domains can also be used.

Exemplary Type IIS restriction enzymes are described in InternationalPublication WO 07/014275, incorporated herein in its entirety.Additional restriction enzymes also contain separable binding andcleavage domains, and these are contemplated by the disclosure. See, forexample, Roberts et al. (2003) Nucleic Acids Res. 31:418-420.

In certain embodiments, the cleavage domain comprises one or moreengineered cleavage half-domain (also referred to as dimerization domainmutants) that minimize or prevent homodimerization, as described, forexample, in U.S. Patent Publication Nos. 20050064474 and 20060188987 andWO 2013/130824. Exemplary engineered cleavage half-domains of Fok I thatform obligate heterodimers include a pair in which a first cleavagehalf-domain includes mutations at amino acid residues at positions 490and 538 of Fok I and a second cleavage half-domain includes mutations atamino acid residues 486 and 499. See, e.g., U.S. Patent Publication No.2008/0131962 and 2011/0201055. Engineered cleavage half-domainsdescribed herein can be prepared using any suitable method, for example,by site-directed mutagenesis of wild-type cleavage half-domains (Fok I)as described in U.S. Patent Publication Nos. 20050064474 and20080131962.

The term “edit”, “edits,” “editing,” and the like refer to any kind ofengineering, altering, modifying or modulating (in each case whichincludes, but not limited to, by means of gene knockout, gene tagging,gene disruption, gene mutation, gene insertion, gene deletion, geneactivation, gene silencing or gene knock-in).

As used herein, “genetic modification,” “genome editing,” “genomemodification,” “gene modification,” and “gene editing,” refer to anygene addition, deletion, knock-out, knock-in, tagging, mutation,activation, silencing, modification, and/or disruption to a cell'snucleotides. The cell in this context can be in vitro, in vivo, or exvivo.

By “target genomic region,” “target gene,” “DNA target”, “DNA targetsequence”, “target sequence”, “target nucleotide sequence”,“target-site”, “target”, “site of interest”, “recognition site”,“polynucleotide recognition site”, “recognition sequence”, “cleavagesite” is intended a polynucleotide sequence that is recognized andcleaved by a genome editing system. These terms refer to a distinct DNAlocation, preferably a genomic location, at which a DNA break (cleavage)is to be induced by the genome editing system.

The aforesaid editing, including engineering, altering, modifying andmodulating, can occur simultaneously or sequentially. Any genome editingsystem known in the art can be used. In some embodiments, the genomeediting system is a meganuclease based system, a zinc finger nuclease(ZFN) based system, a Transcription Activator-Like Effector-basedNuclease (TALEN) based system, a CRISPR-based system, or NgAgo-basedsystem.

Meganuclease-based, ZFN-based and TALEN-based each comprise at least oneDNA-binding domain or a nucleic acid comprising a nucleic acidsequence(s) encoding the DNA-binding domain, and achieve specifictargeting or recognition of a target genomic region(s) via protein-DNAinteractions. A CRISPR-based system comprises at least one guide RNAelement or a nucleic acid comprising a nucleic acid sequence(s) encodingthe guide RNA element, and achieves specific targeting or recognition ofa target genomic region(s) via base-pairs directly with the DNA of thetarget genomic region(s). A NgAgo-based system comprises at least oneguide DNA element or a nucleic acid comprising a nucleic acidsequence(s) encoding the guide DNA element, and achieves specifictargeting or recognition of a target genomic region(s) via base-pairsdirectly with the DNA of the target genomic region(s).

In some embodiments, the genome editing system is a meganuclease-basedsystem. A meganuclease-based system employs meganucleases which areendonucleases with large (>14bp) recognition sites, and its DNA bindingdomains are also responsible for cleavage of target sequences. TheDNA-binding domain of meganucleases may have a double-stranded DNAtarget sequence of 12 to 45 bp. In some embodiments, the meganuclease iseither a dimeric enzyme, wherein each meganuclease domain is on amonomer, or a monomeric enzyme comprising the two domains on a singlepolypeptide. Not only wild-type meganucleases but also variousmeganuclease variants have been generated by protein engineering tocover a myriad of unique sequence combinations. In some embodiments,chimeric meganucleases with a recognition site composed of a half-siteof meganuclease A and a half-site of protein B can also be used.Specific examples of such chimeric meganucleases compriaing the proteindomains of I-DmoI and I-CreI. Examples of meganucleases include homingendonucleases from the LAGLIDADG family

The LAGLIDADG meganuclease can be I-SceI, I-ChuI, I-CreI, I-CsmI,PI-SceI, PI-TliI, PI-MtuI, I-CeuI, I-SceII, I-SceIII, HO, PI-CivI,PI-CtrI, PI-AaeI, PI-BsuI, PI-DhaI, PI-DraI, PI-MavI, PI-MchI, PI-MfuI,PI-MflI, PI-MgaI, PI-MgoI, PI-MinI, PI-MleI, PI-MuraI, PI-MshI, PI-MsmI,PI-MthI, PI-MtuI, PI-MxeI, PI-NpuI, PI-PfuI, PI-RmaI, PI-SpbI, PI-SspI,PI-FacI, PI-MjaI, PI-PhoI, PI-TagI, PI-ThyI, PI-TkoI, PI-TspI, orI-MsoI; or can be a functional mutant or variant thereof, whetherhomodimeric, heterodimeric or monomeric. In some embodiments, theLAGLIDADG meganuclease is a I-CreI derivative. In some embodiments, theLAGLIDADG meganuclease shares at least 80% similarity with the naturalI-CreI LAGLIDADG meganuclease. In some embodiments, the LAGLIDADGmeganuclease shares at least 80% similarity with residues 1-152 of thenatural I-CreI LAGLIDADG meganuclease. In some embodiments, theLAGLIDADG meganuclease may consists of two monomers sharing at least 80%similarity with residues 1-152 of the natural I-CreI LAGLIDADGmeganuclease linked together, with or without a linker peptide.

The “LAGLIDADG meganuclease” refers to a homing endonuclease from theLAGLIDADG family, as defined in Stoddard et al (Stoddard, 2005), or anengineered variant comprising a polypeptide sharing at least 80%, 85%,90%, 95%, 97.5%, 99% or more identity or similarity with said naturalhoming endonuclease. Such engineered LAGLIDADG meganucleases can bederived from monomeric or dimeric meganucleases. When derived fromdimeric meganucleases, such engineered LAGLIDADG meganucleases can besingle-chain or dimeric endonucleases.

By “I-CreI” is intended the natural wild-type I-CreI meganuclease havingthe sequence of pdb accession code 1g9y.

The DNA recognition and cleavage functions of meganucleases aregenerally intertwined in a single domain. Unlike meganulceases, the DNAbinding domains of ZFN-based and TALEN-based systems are distinct fromthe endonuclease for cleavage function. The ZFN-based system comprises:at least one zinc finger protein or a variant thereof, or a nucleic acidcomprising a nucleotide sequence(s) encoding the zinc finer protein orvariant thereof as its DNA-binding domain; and an endonuclease element,such as zinc finger nuclease (ZFN) or FokI cleavage domain. The zincfinder protein (ZFP) is non-naturally occurring in that it is engineeredto bind to a target site of choice. See, for example, Beerli et al.(2002) Nature Biotechnol. 20: 135-141; Pabo et al. (2001) Ann. Rev.Biochem. 70:313-340; Isalan ei al. (2001) Nature Biotechnol. 19:656-660;Segal et al. (2001) Curr. Opin. Biotechnol. 12:632-637; Choo et al.(2000) Curr. Opin. Struct Biol. 10:411-416; U.S. Pat. Nos. 6,453,242;6,534,261; 6,599,692; 6,503,717; 6,689,558; 7,030,215; 6,794,136;7,067,317; 7,262,054; 7,070,934; 7,361,635; 7,253,273; and U.S. PatentPublication Nos. 2005/0064474; 2007/0218528; 2005/0267061.

An engineered zinc finger binding domain can have a novel bindingspecificity, compared to a naturally-occurring zinc finger protein.Engineering methods include, but are not limited to, rational design andvarious types of selection. Rational design includes, for example, usingdatabases comprising triplet (or quadruplet) nucleotide sequences andindividual zinc finger amino acid sequences, in which each triplet orquadruplet nucleotide sequence is associated with one or more amino acidsequences of zinc fingers which bind the particular triplet orquadruplet sequence. See, for example, U.S. Patents 6,453,242 and6,534,261, incorporated by reference herein in their entireties.

Various kinds of selection methods can be used with the methods herein.Exemplary selection methods, including phage display and two-hybridsystems, are disclosed in U.S. Pat. Nos. 5,789,538; 5,925,523;6,007,988; 6,013,453; 6,410,248; 6,140,466; 6,200,759; and 6,242,568; aswell as WO 98/37186; WO 98/53057; WO 00/27878; WO 01/88197 and GB2,338,237. In addition, enhancement of binding specificity for zincfinger binding domains has been described, for example, in WO02/077227.In addition, as disclosed in these and other references, zincfinger domains and/or multi-fingered zinc finger proteins may be linkedtogether using any suitable linker sequences, including for example,linkers of 5 or more amino acids in length. See, also, U.S. Pat. Nos.6,479,626; 6,903,185; and 7,153,949 for exemplary linker sequences 6 ormore amino acids in length. The proteins described herein may includeany combination of suitable linkers between the individual zinc fingersof the protein. Selection of target sites; ZFPs and methods for designand construction of fusion proteins (and polynucleotides encoding same)are known to those of skill in the art and described in detail in U.S.Pat. Nos. 6,140,0815; 789,538; 6,453,242; 6,534,261 ; 5,925,523;6,007,988; 6,013,453; 6,200,759; WO 95/19431; WO 96/06166; WO 98/53057;WO 98/54311; WO 00/27878; WO 01/60970 WO 01/88197; WO 02/099084; WO98/53058; WO 98/53059; WO 98/53060; WO 02/016536 and WO 03/016496.

In addition, as disclosed in these and other references, zinc fingerdomains and/or multi-fingered zinc finger proteins may be linkedtogether using any suitable linker sequences, including for example,linkers of 5 or more amino acids in length. See, also, U.S. Pat. Nos.6,479,626; 6,903,185; and 7,153,949 for exemplary linker sequences 6 ormore amino acids in length. The proteins described herein may includeany combination of suitable linkers between the individual zinc fingersof the protein.

A Transcription Activator-Like Effector-based Nuclease (TALEN) systemrefers to a genome editing system that employs one or more TranscriptionActivator-Like Effector (TALE)-DNA binding domain and an endonucleaseelement, such as FokI cleavage domain. The TALE-DNA binding domaincomprises one or more TALE repeat units, each having 30-38 (such as, 31,32, 33, 34, 35, or 36) amino acids in length. The TALE-DNA bindingdomain may employ a full length TALE protein or fragment thereof, or avariant thereof. The TALE-DNA binding domain can be fused or linked tothe endonuclease domain by a linker.

The terms “CRISPR-based system, ” “CRISPR-based gene editing system,”“CRISPR-genome editing,” “CRISPR-gene editing,” “CRISPR-endonucleasebased genome editing,” and the like are used interchangeably herein, andcollectively refer to a genome editing system that comprises one or moreguide RNA elements; and one or more RNA-guided endonuclease elements.The guide RNA element comprises a targeter RNA comprising a nucleotidesequence substantially complementary to a nucleotide sequence at the oneor more target genomic regions or a nucleic acid comprising a nucleotidesequence(s) encoding the targeter RNA. The RNA-guided endonucleaseelement comprises an endonuclease that is guided or brought to a targetgenomic region(s) by a guide RNA element; or a nucleic acid comprising anucleotide sequence(s) encoding such endonuclease. Examples of suchCRISPR-based gene editing system includes CRISPR-based system is aCRISPR-Cas system or a CRISPR-Cpf system.

As used herein, the terms “guide RNA element,” “guide RNA”, “gRNA,”“gRNA molecule,” and “synthetic guide RNA” are used interchangeably andrefer to the polynucleotide sequence comprising a targeter RNA thathybridizes with a target nucleic sequence or a nucleic acid comprising anucleotide sequence(s) encoding the targeter RNA. A targeter RNA of gRNAcomprises a targeting domain that includes a nucleotide sequencesubstantially complementary to the nucleotide sequence at a targetgenomic region. The phrase “substantially complementary” means a degreeof complementarity that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%.95%, 97%, 98%, 99%, or 100% over a region of 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, or morenucleotides, or refers to two nucleic acids that hybridize understringent conditions.

A guide RNA element can further comprise an activator RNA that iscapable of hybridizing with the targeter RNA, or a nucleic acidcomprising a nucleotide sequence(s) encoding the activator RNA. Theactivator RNA and targeter RNA can be separate or fused as a singlenucleic acid via a linker loop sequence to form a single gRNA molecule.A gRNA molecule may comprise a number of domains. For example, such gRNAcomprises, for example from 5′ to 3′: a targeting domain (which iscomplementary to a target nucleic acid); a first complementarity domain;a linking domain; a second complementarity domain (which iscomplementary to the first complementarity domain); a proximal domain;and a optionally, a tail domain. See WO2015048557.

A “first complementarity domain” has substantial complementarity withthe second complementarity domain, and may form a duplexed region underat least some physiological conditions.

A “linking domain” serves to link the first complementarity domain withthe second complementarity domain of a unimolecular gRNA. The linkingdomain can link the first and the second complementarity domainscovalently or non-covalently.

A “proximal domain” can be 3-25 nucleotides in length, or 5-20nucleotides in length. The proximal domain can share homology with or bederived from a naturally occurring proximal domain.

A “tail domain” can be absent, or be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10nucleotides in length. The tail domain may include sequences that arecomplemtary to each other and which, under at least some physiologicalconditions, form a duplexed region.

The guide RNA element may form a complex with an endonuclease of theRNA-guided endonuclease element, such as Cas endonuclease(“gRNA/nuclease complex”). An example of gRNA/nuclease complex is aCRISPR complex as described below with respect to a CRISR-based system.In some embodiments, the CRISPR complex comprises an endonuclease ofRNA-guided endonuclease system that is complexed with the targeter RNA.In some embodiments, the CRISPR complex comprises an endonuclease ofRNA-guided endonuclease system that is complexed with the targeter RNAand the activator RNA.

The targeting domain of targeter RNA promotes specific targeting orhoming of a gRNA/nuclease complex to a target nucleotide sequence. Insome embodiments, the targeting domain can be 10-30 bp, such as 15-25bp, 18-22 bp, or 20 bp.

Methods for designing gRNAs are known in the art, including methods forselecting, designing, and validating target domain. See, for example,WO2015048577, Mali et al., 2013 SCIENCE 339(6121): 823-826; Hsu et al.,2013 NATBIOTECHNOL, 31(9): 827-32; Fu et al., 2014 NATBTOTECHNOL, doi:10.1038/nbt.2808. PubMed PMID: 24463574; Heigwer et al., 2014 NATMETHODS 11 (2): 122-3. doi: 1 0.1038/nmeth.2812. PubMed PMID: 24481216;Bae et al., 2014 BIOTNFORMATICS PubMed PMID: 24463181; Xiao A et al.,2014 BIOINFORMATICS Pub Med PMID: 24389662.

In some embodiments, RNA-guided endonucleases, such as a Cas enzyme orprotein (e.g., Type-II Cas9 protein) or Cpf enzyme or protein (e.g.,Cpf1 protein) can be used. In some embodiments, a modified version ofsuch Cas or Cpf enzyme or protein can also be used.

In some embodiments, the CRISPR-based system is a CRISPR-Cas system. TheCRISPR-Cas system comprises: (a) at least one guide RNA element or anucleic acid comprising a nucleotide sequence(s) encoding the guide RNAelement, the guide RNA element comprising a targeter RNA that includes anucleotide sequence substantially complementary to a nucleotide sequenceat the one or more target genomic regions, and an activator RNA thatincludes a nucleotide sequence that is capable of hybridizing with thetargeter RNA; and (b) a Cas protein element comprising a Cas protein ora nucleic acid comprising a nucleotide sequence encoding the Casprotein. The targeter RNA and activator RNAs can be separate or fusedtogether into a single RNA.

In some embodiments, the CRISPR-based system includes Class 1 CRISPRand/or Class 2 CRISPR systems. Class 1 systems employ several Casproteins together with a CRISPR RNAs (crRNA) as the targeter RNA tobuild a functional endonuclease. Class 2 CRISPR systems employ a singleCas protein and a crRNA as the targeter RNA. Class 2 CRISPR systems,including the type II Cas9-based system, comprise a single Cas proteinto mediate cleavage rather than the multi-subunit complex employed byClass I systems. The CRISPR-based system also includes Class II, Type VCRISPR system employing a Cpf1 protein and a crRNA as the targeter RNA.

The Cas protein is a CRISPR-associated (Cas) double stranded nuclease.In some embodiments, CRISPR-Cas system comprises a Cas9 protein. In someembodiments, the Cas9 protein is SaCas9, SpCas9, SpCas9n, Cas9-HF,Cas9-H840A, FokI-dCas9, or D10A nickase. The term “Cas protein,” such asCas9 protein, include wild-type Cas protein or functional derivativesthereof (such as truncated versions or variants of the wild-type Casprotein with a nuclease activity).

In some embodiments, Cas9 proteins from species other than S. pyogenesand S. thermophiles can be used. Additional Cas9 protein species may beobtained and used herein include: Acidovorax avenae, Actinobacilluspleuropneumonias, Actinobacillus succinogenes, Actinobacillus suis,Actinomyces sp., cycliphilus denitrificans, Aminomonas paucivorans,Bacillus cereus; Bacillus smithii, Bacillus thuringiensis, Bacteroidessp., Blastopirellula marina, Bradyrhizobium sp., Brevibacilluslaterosporus, Campylobacter coli, Campylobacter jejuni, Campylobacterlari, Candidatus Puniceispirillum, Clostridium cellulolyticum,Clostridium perfingens, Corynebacterium accolens, Corynebacteriumdolichum, Corynebacterium matruchotii, Dinoroseobacter shibae,Eubacterium dolichum, gamma proteobacterium, Gluconacetobacterdiazotrophicus, Haemoplzilus parainfluenzae, Haemophilus sputorum,Helicobacter canadensis, Helicohacter cinaedi, Helicobacter mustelae,llyobacter polytropus, Kingella kingae, lactobacillus crispatus,listeria ivanovii, listeria monocytogenes, listeriaceae bacterium,Methylocystis sp.,Methylosinus trichosporium, Mobiluncus mulieris,Neisseria bacilliformis, Neisseria cinerea, Neisseria flavescens,Neisseria lactamica, Neisseria sp., Neisseria wadsworthii, Nitrosomonassp., Parvibaculum lavamentivorans, Pasteurella multocida,Phascolarctobacterium succinatutells, Ralstonia syzygii,Rhodopseudomonas palustris, Rhodovulum sp., Simonsiella muelleri,Sphingomonas sp., Sporolactobacillus vineae, Staphylococcus lugdunensis,Streptococcus sp., Subdoligranulum sp., Tistrella mobilis, Treponemasp., or Verminephrobacter eiseniae.

In some embodiments, one or more elements of a CRISPR-based system isderived from a type I, type II, or type III CRISPR system

In some embodiments, one or more elements of a CRISPR-based system isderived from a particular organism comprising an endogenous CRISPRsystem, such as Streptococcus pyogenes, Staphylococcus aureus,Francisella tularensis, Prevotella sp., Acidaminococcus sp., andLachnospiraceae sp. In general, a CRISPR-based system is characterizedby elements that promote the formation of a CRISPR complex at the targetgenomic regions or the site of a target sequence (also referred to as aprotospacer in the context of an endogenous CRISPR system). In thecontext of formation of a CRISPR complex, “target sequence” refers to asequence to which a guide sequence is designed to have substantialcomplementarity, where hybridization between a target sequence and aguide sequence promotes the formation of a CRISPR complex. Fullcomplementarity is not necessarily required, provided there issufficient complementarity to cause hybridization and promote formationof a CRISPR complex. A target sequence may comprise any polynucleotide,such as DNA or RNA polynucleotides. In some embodiments, a targetsequence is located in the nucleus or cytoplasm of a cell(s). In someembodiments, the target sequence may be within an organelle of aeukaryotic cell(s), for example, mitochondrion or chloroplast.

A sequence or template that may be used for recombination into thetargeted locus comprising the target sequences is referred to as an“editing template” or “editing polynucleotide” or “editing sequence”. Anexogenous template polynucleotide may be referred to as an editingtemplate or donor template. In some embodiments, single stranded DNA anddouble stranded DNA from either synthetic or biologic origin may beused. By way of non-limiting example, suitable editing templates includessODN, dsODN, PCR products, plasmids, and viruses including AAV,Adenovirus, Retrovirus, lentivirus, etc. Additional editing templatesare also possible. In some embodiments, the recombination is homologousrecombination.

In some embodiments, the CRISPR-based system is a CRISPR-Cas9 system.The targeter RNA of the CRISPR-Cas9 system comprises a CRISPR targetingRNA (crRNA) and the activator RNA of the CRISPR-Cas 9 system comprises atrans-activating CRISPR RNA (tracRNA). The Cas protein element of theCRISPR-Cas9 system employs a Cas9 protein. The crRNA and the tracrRNAcan be separate or combined into a single RNA construct via a linkerloop sequence. This combined RNA construct is called a single-guide RNA(sgRNA; or guide RNA).

With respect to general information on CRISPR-Cas systems, componentsthereof, and delivery of such components, including methods, materials,delivery vehicles, vectors, particles, AAV, and making and usingthereof, including as to amounts and formulationscan be found in: U.S.Pat. Nos. 8,999,641, 8,993,233, 8,945,839, 8,932,814, 8,906,616,8,895,308, 8,889,418, 8,889,356, 8,871,445, 8,865,406, 8,795,965,8,771,945 and 8,697,359; US Patent Publications US 2014-0310830, US2014-0287938 A1, US 2014-0273234 A1, U52014-0273232 A1, US 2014-0273231,US 2014-0256046 A1, US 2014-0248702 A1, US 2014-0242700 A1, US2014-0242699 A1, US 2014-0242664 A1, US 2014-0234972 A1, US 2014-0227787A1, US 2014-0189896 A1, US 2014-0186958, US 2014-0186919 A1, US2014-0186843 A1, US 2014-0179770 Al and US 2014-0179006 A1, US2014-0170753; European Patents EP 2 784 162 B1 and EP 2 771 468 Bl;European Patent Applications EP 2 771 468 (EP13818570.7), EP 2 764 103(EP13824232.6), and EP 2 784 162 (EP14170383.5); and PCT PatentPublications PCT Patent Publications WO 2014/093661, WO 2014/093694, WO2014/093595, WO 2014/093718, WO 2014/093709, WO 2014/093622, WO2014/093635, WO 2014/093655, WO 2014/093712, WO2014/093701,WO2014/018423, WO 2014/204723, WO 2014/204724, WO 2014/204725, WO2014/204726, WO 2014/204727, WO 2014/204728, WO 2014/204729, andWO2016/028682.

In some embodiments, the CRISPR-based system is a CRISPR-Cpf system. The“CRISPR-Cpf system” comprises: (a) at least one guide RNA element or anucleic acid comprising a nucleotide sequence(s) encoding the guide RNAelement, the guide RNA comprising a targeter RNA having a nucleotidesequence complementary to a nucleotide sequence at a locus of the targetnucleic acid; and (b) a Cpf protein element or a nucleic acid comprisinga nucleotide sequence encoding the Cpf protein element.

An example of a Cpf protein element includes a Cpf1 nucleases, such asFrancisella Cpf1 (FnCpf1) and any variants thereof. See, for example,Zetsche et al., “Cpf1 is a single RNA-guided endonuclease of a class 2CRISPR-Cas system,” Cell, 163(3): pages 759-71; and Fonfara et al., “TheCRISPR-associated DNA-cleaving enzyme Cpf1 also processes precursorCRISPR RNA,” Nature 532 (7600): pages, 517-21. Cpf1's preferred PAM is5′-TTN, differing from that of Cas9 (3′ -NGG) in both genomic locationand GC-content. The CRISPR-Cpf system may not employ an activator RNA(tracrRNA). Both Cpf1 and its guide RNAs are in general smaller thantheir SpCas9 counterparts. The Cpf1 locus contains a mixed alpha/betadomain, a RuvC-I followed by a helical region, a RuvC-II and a zincfinger-like domain. The Cpf1 protein has a RuvC-like endonuclease domainthat is similar to the RuvC domain of Cas9. Furthermore, Cpf1 does nothave a HNH endonuclease domain, and the N-terminal of Cpf1 does not havethe alfa-helical recognition lobe of Cas9. The Cpf1 loci encode Cas1,Cas2 and Cas4 proteins more similar to types I and III than from type IIsystems. Cpf1-family proteins can be found in many bacterial species.

Without it being hound to a particular theory, the CRISPR-Cpf systememploys a Cpf1-crRNA complex which cleaves target DNA or RNA byidentification of a protospacer adjacent motif 5′-YTN-3′-(where “Y” is apyrimidine and “N” is any nucleobase) or 5′-TTN-3 in contrast to theG-rich PAM targeted by Cas9. After identification of PAM, Cpf1introduces a sticky-end-like DNA double-stranded break of 4 or 5nucleotides overhang.

In some embodiments, the genome editing system is a NgAgo-based system.The NgAgo-based system comprises at least one guide DNA element or anucleic acid comprising a nucleic acid sequence(s) encoding the guideDNA element; and a DNA-guided endonuclease. The NgAgo-based systememploys DNA as a guide element. Its working principle is similar to thatof CRISPR-Cas9 technology, but its guide element is a segment of guideDNA (dDNA) rather than gRNA in CRISPR-Cas9 technology. An example ofDNA-guided endonuclease is an Argonaute endonuclease (NgAgo) fromNatronobacterium gregoryi. See, for example, Feng Gao et al. “DNA-guidedgenome editing using the Natronobacterium gregoryi Argonaute,” NatureBiotechnology, (2016): doi:10.1038/nbt.3547.

By “linker,” “peptide linker”, “peptidic linker” or “peptide spacer” itis intended to mean a peptide sequence that allows the connection ofdifferent monomers in a fusion protein and the adoption of the correctconformation for said fusion protein activity and which does not alterthe activity of either of the monomers. Peptide linkers can be ofvarious sizes from 1, 2, 3, 4, 5, 10, 15, 20, 30, 40 to 50 amino acidsas a non limiting indicative range or any intermediate value within thisrange.

DNA-PK Inhibitors for Increasing Genomic Editing Efficiency

Targeted genome editing efficiency can be increased by administering toa cell(s) with one or more compounds (e.g., DNA-PK inhibitors) describedherein and a genome editing system. Genome editing systems suitable foruse include, for example, a meganuclease based system, a zinc fingernuclease (ZFN) based system, a Transcription Activator-LikeEffector-based Nuclease (TALEN) system, a CRISPR-based system orNgAgo-based system. The methods, compositions, and kits of thedisclosure provide DNA-PK inhibitors and/or a genome editing system forincreasing genome editing efficiency. In some embodiments, HDR genomeediting efficiency is increased following administering to a cell(s)with a DNA-PK inhibitor.

In some embodiments, the genome editing system is a CRISPR-based genomeediting system. The CRISPR-based genome editing system can be aCRISPR-Cas system or variants thereof. The CRISPR-Cas system can use anyCas endonucleases, such as Cas 9 endonucleases and variants thereof.Examples of Cas 9 endonucleases includes Cas9 endonucleases or variantsthereof, such as SaCas9, SpCas9, SpCas9n, Cas9-HF, Cas9-H840A,FokI-dCas9, or CasD10A nickase. The Cas endonuclease can be wild type,engineered, or a nickase mutant, or any variations thereof.

In some embodiments, the CRISPR-based genome editing system includes aCRISPR sequence, a trans-activating cr (tracr) sequence, a guidesequence and a Cas endonuclease or any combinations thereof.

In some embodiments, the CRISPR-based genome editing system includes aRNA comprising a CRISPR sequence (crRNA), a RNA comprising atrans-activating cr (tracr) sequence (tracrRNA) and a Cas endonucleaseor any combinations thereof.

In some embodiments, the CRISPR-based genome editing system includes aCRISPR sequence sequence, a guide sequence, and a Cas endonuclease or aCpf endonuclease, or any combinations thereof.

In some embodiments, the CRISPR-based genome editing system is aCRISPR-Cpf system. The Cpf nuclease is a Class 2 CRISPR-Cas systemendonuclease. Cpf is a single RNA-guided endonuclease. The Cpf nucleasecan be wild type, engineered or a nickase mutant, or any variationsthereof. See, for example, Zetsche et al., “CPF1 is a single RNA-guidedendonuclease of a Class 2 CRISPR-Cas System,” Cell, 163(3): 759-71. Insome embodiments, the Cpf nuclease is a Cpf 1 endonuclease.

In some embodimentss, the genome editing system is a meganuclease basedsystem. Meganuclease-based genome editing uses sequence-specificendonucleases that recognize large DNA target sites (e.g. typicallyabout >12bp). See, for example, U.S. Pat. No. 9,365,964. Meganucleasescan cleave unique chromosomal sequences without affecting overall genomeintegrity. In some embodiments, the meganuclease can be a homingendonuclease. In some embodiments, the meganuclease can be an intronendonuclease or an intein endonuclease. The homing endonucleases canbelong to the LAGLIDADG family The meganucleases can be wild type,engineered or a nickase mutant.

In some embodimentss, the gene-editing system is a zinc finger nuclease(ZFN) based system. The ZFN is an artificial restriction enzymeengineered based on the fusion between a zing finger DNA-binding domainand a DNA-cleavage domain. See, for example, U.S. Pat. No. 9,145,565.

In some embodiments, the gene-editing system is a TranscriptionActivator-Like Effector-based Nuclease (TALEN). TALENs are engineeredrestriction enzymes that are made by the fusion of a TAL effectorDNA-binding domain to a DNA cleavage domain. See, for example, U.S. Pat.No. 9,181,535.

In some embodiments, the gene editing system is an Argonaute basedsystem. Argonaute based gene editing systems include an Argonautederived endonuclease and a 5′ phosphorylated ssDNA. In some embodiments,the phosphorylated ssDNA can be 10-40 nucleotides, 15-30 nucleotide or18-30 nucleotides (e.g, about 24 nucleotides) in length. In someembodiments, the Argonaute endonuclease can be any endonuclease. In someembodiments, the Argonaute endonuclease is derived from Thermusthermophiles (TtAgo), Pyrococcus furiosus (PfAgo), or Natronobacteriumgregoryi (NgAgo). In some embodiments, the Natrobacterium gregoryi(NgAgo) is strain 2 (i.e. N. gregoryi SP2). In some embodiments, theArgonaute endonuclease is NgAgo. See, for example, Gao et al.,“DNA-guided genome editing using the Natronobacterium gregoryiArgonaute,” Nature Biotechnology, May 2016.

The DNA-PK inhibitors can be any DNA-PK inhibitor. The DNA-PK inhibitorcan be any compound or substance that causes inhibition of a DNA-PK. TheDNA-PK inhibitor can be a compound, small molecule, antibody, ornucleotide sequence. In some embodiments, the DNA-PK inhibitors arecompounds represented by Formula (III-E-1) or (III-E-2).

In some embodiments, the disclosure provides a method of editing one ormore target genomic regions, the method includes administering to one ormore cells that have one or more target genomic regions, a genomeediting system and a compound represented by Formula (III-E-1) or(III-E-2):

wherein:

X is O or NR; wherein R is H or C₁C₄ alkyl;

Y is O, or NR; wherein R is H or C₁-C₄ alkyl;

R³ is hydrogen, C₁₋₄ alkyl, or OC₁₋₂ alkyl;

R¹ is a 6-membered heteroaromatic ring containing one or two nitrogenatoms wherein the heteroaromatic ring may be substituted by 0, 1, or 2substituents R² independently selected from the group consisting ofC₁-C₄-alkyl, C₁-C₄-haloalkyl, C(═O)NHR²′, and NR⁴R⁵; wherein

R²′ is C₁-C₄ alkyl,

each R⁴ and R⁵ is independently H, C₁-C₄ alkyl, or C(═O)C₁-C₄ alkyl; or

R⁴ and R⁵ together with the N atom to which they are attached form aheterocyclic ring comprising 0 or 1 additional N atom wherein saidheterocyclic ring may be substituted by C₁-C₄ alkyl; and

Ring B is selected from the group consisting of:

wherein W is N or CR³; and Z is O or S; wherein R³ is H or C₁-C₄ alkyl.

In some embodiments, the disclosure provides a method of editing one ormore target genomic regions, the method includes administering to one ormore cells that have one or more target genomic regions, a genomeediting system and a compound represented by Formula (III-E-1) or(III-E-2),or pharmaceutically acceptable salts thereof.

In some embodiments, the disclosure also provides a method of repairinga DNA break in one or more target genomic regions via a homologydirected repair (HDR) pathway, the method includes administering to oneor more cells that have one or more target genomic regions, a genomeediting system and a compound represented by Formula (III-E-1) or(III-E-2)), or pharmaceutically acceptable salts thereof.

The genome editing system interacts with a nucleic acid(s) of the targetgenomic regions, resulting in a DNA break, and wherein the DNA break isrepaired at least in part via a HDR pathway.

In some embodiments, the disclosure also provides a method of inhibitingor suppressing repair of a DNA break in one or more target genomicregions via a NHEJ pathway, the method includes administering to one ormore cells that have one or more target genomic regions, a genomeediting system and a compound represented by Formula (III-E-1) or(III-E-2)), or pharmaceutically acceptable salts thereof.

The the genome editing system interacts with a nucleic acid(s) of theone or more target genomic regions, resulting in a DNA break, andwherein repair of the DNA break via a NHEJ pathway is inhibited orsuppressed.

In some embodiments, the disclosure also provides a method of modifyingexpression of one or more genes or proteins, the method includesadministering to one or more cells that comprise one or more targetgenomic regions, a genome editing system and a compound represented byFormula (III-E-1) or (III-E-2), or pharmaceutically acceptable saltsthereof.

The genome editing system interacts with a nucleic acid(s) of the one ormore target genomic regions of a target gene(s), resulting in editingthe one or more target genomic regions and wherein the edit modifiesexpression of a downstream gene (s) and/or protein(s) associated withthe target gene(s).

In some embodiments, the DNA break includes a DNA double strand break(DSB).

In some embodiments, the efficiency of the repair of the DNA break atthe target genomic regions in the one or more cells via a HDR pathway isincreased as compared to that in otherwise identical cell or cells butwithout the compound.

In some embodiments, the efficiency of inhibiting or suppressing therepair of the DNA break at the target genomic regions in the one or morecells via a NHEJ pathway is increased as compared to that in otherwiseidentical cell or cells but without the compound.

In some embodiments, the efficiency is increased by at least 2-fold,3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold,40-fold, 50-fold, or 100-fold as compared to that in otherwise identicalcell or cells but without compound.

In some embodiments, the efficiency is measured by frequency of targetedpolynucleotide integration. In some embodiments, the efficiency ismeasured by frequency of targeted mutagenesis. In some embodiments, thetargeted mutagenesis comprises point mutations, deletions, and/orinsertions.

In some embodiments, the expression of a downstream gene (s) and/orprotein(s) associated with the target gene(s) is increased as comparedto the baseline expression level in the one or more cells prior to theadministration. For example, said expression is increased by at least10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1-fold, 1.5-fold, 2-fold,2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, or 10-fold ascompared to the baseline expression level in the one or more cells priorto the administration.

In some embodiments, the expression of a downstream gene (s) and/orprotein(s) associated with the target gene(s) is decreased as comparedto the baseline expression level in the one or more cells prior to theadministration. For example, the gene expression is decreased by atleast 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%,or 99% as compared to the baseline expression level in the one or morecells prior to the administration.

In some embodiments, the expression of a downstream gene (s) and/orprotein(s) associated with the target gene(s) is substantiallyeliminated in the one or more cells.

In some embodiments, the cell is synchronized at the S or the G2 cellcycle phase.

In some embodiments, the one or more cells that are administered orcontacted with said compound have increased survival in comparison toone or more cells that have not been administered or contacted with saidcompound.

In some embodiments, the genome editing system and the compound areadministered into the one or more cells simultaneously. In someembodiments, the genome editing system and the compound are administeredinto the one or more cells sequentially. In some embodiments, the genomeediting system is administered into the one or more cells prior to thecompound. In some embodiments, the compound is administered into the oneor more cells prior to the genome editing system.

In some embodiments, the one or more cells are cultured cells. In someembodiments, the one or more cells are in vivo cells within an organism.In some embodiments, the one or more cells are ex vivo cells from anorganism.

In some embodiments, the organism is a mammal. In some embodiments, theorganism is a human.

In some embodiments, the genome editing system and the compound areadministered via a same route. In some embodiments, the genome editingsystem and the compound are administered via a different route. In someembodiments, the genome editing system is administered intravenously andthe compound is administered orally.

In some embodiments, the genome editing system is selected from ameganuclease based system, a zinc finger nuclease (ZFN) based system, aTranscription Activator-Like Effector-based Nuclease (TALEN) system, aCRISPR-based system, or a NgAgo-based system.

In some embodiments, the genome editing system is a CRISPR-based system.In some embodiments, the CRISPR-based system is a CRISPR-Cas system or aCRISPR-Cpf system.

In some embodiments, the CRISPR-based system is a CRISPR-Cas system andwherein the CRISPR-Cas system includes: (a) at least one guide RNAelement that includes: (i) a targeter RNA that includes a nucleotidesequence substantially complementary to a nucleotide sequence at the oneor more target genomic regions or a nucleic acid that includes anucleotide sequence(s) encoding the targeter RNA; (ii) and an activatorRNA that includes a nucleotide sequence that is capable of hybridizingwith the targeter RNA or a nucleic acid that includes a nucleotidesequence(s) encoding the activator RNA; and (b) a Cas protein elementthat includes a Cas protein or a nucleic acid that includes a nucleotidesequence(s) encoding the Cas protein.

In some embodiments, the targeter RNA and activator RNA are fused as asingle molecule.

In some embodiments, the Cas protein is a Type-II Cas9 protein. In someembodiments, the Cas9 protein is a SaCas9, SpCas9, SpCas9n, Cas9-HF,Cas9-H840A, FokI-dCas9, or D10A nickase, or any combinations thereof.

In some embodiments, the CRISPR-based system is a CRISPR-Cpf system andthe CRISPR-Cpf system includes: (a) at least one guide RNA element or anucleic acid that includes a nucleotide sequence(s) encoding the guideRNA element, the guide RNA that includes a targeter RNA that thatincludes a nucleotide sequence substantially complementary to anucleotide sequence at the one or more target genomic regions; and (b) aCpf protein element that includes a Cpf protein or a nucleic acidcomprising a nucleotide sequence encoding the Cpf protein.

In some embodiments, the genome editing system is delivered by one ormore vectors.

In some embodiments, the one or more vectors are selected from viralvectors, plasmids, or ssDNAs.

In some embodiments, the viral vectors are selected from retroviral,lentiviral, adenoviral, adeno-associated and herpes simplex viralvectors.

In some embodiments, the genome editing system is delivered by syntheticRNA.

In some embodiments, the genome editing system is delivered by ananoformulation.

In some embodiments, a kit or composition is provided for editing one ormore target genomic regions. In some embodiments ,the kit or compositionincludes a genome editing system; and a compound represented by formula(III-E-1) or (III-E-2), or pharmaceutically acceptable salts thereof.

In some embodiments, the genome editing system of the kit or compositionis a meganuclease based system, a zinc finger nuclease (ZFN) basedsystem, a Transcription Activator-Like Effector-based Nuclease (TALEN)system, a CRISPR-based system, or NgAgo-based system. In someembodiments, the genome editing system of the kit or composition is aCRISPR-based system. In some embodiments, the CRISPR-based system of thekit or composition is a CRISPR-Cas system or a CRISPR-Cpf system.

In some embodiments, the CRISPR-based system of the kit or compositionis a CRISPR-Cas system and wherein the CRISPR-Cas system includes: (a)at least one guide RNA element that includes: (i) a targeter RNA thatincludes a nucleotide sequence substantially complementary to anucleotide sequence at the one or more target genomic regions or anucleic acid that includes a nucleotide sequence(s) encoding thetargeter RNA; (ii) and an activator RNA that includes a nucleotidesequence that is capable of hybridizing with the targeter RNA, or anucleic acid that includes a nucleotide sequence(s) encoding theactivator RNA; and (b) a Cas protein element that includes a Cas proteinor a nucleic acid that includes a nucleotide sequence(s) encoding theCas protein.

In some embodiments, the Cas protein of the kit or composition is aType-II Cas9 protein. In some embodiments, the Cas9 protein of the kitor composition is a SaCas9, SpCas9, SpCas9n, Cas9-HF, Cas9-H840A,FokI-dCas9, or D10A nickase, or any combination thereof.

In some embodiments, the CRISPR-based system of the kit or compositionis a CRISPR-Cpf system, and wherein the CRISPR-Cpf system includes: (a)a targeter RNA that includes a nucleotide sequence substantiallycomplementary to a nucleotide sequence at the one or more target genomicregions, or a nucleic acid that includes a nucleotide sequence(s)encoding the targeter RNA; and (b) a Cpf protein element that includes aCpf protein or a nucleic acid that includes a nucleotide sequence(s)encoding the Cpf protein.

In some embodiments, the genome editing system of the kit or compositionis included or packaged in one or more vectors. In some embodiments, theone or more vectors are selected from viral vectors, plasmids, orssDNAs. In some embodiments, the viral vectors are selected from thegroup consisting of retroviral, lentiviral, adenoviral, adeno-associatedand herpes simplex viral vectors.

In some embodiments, the increased genome editing efficiency is about1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold,25-fold, 30-fold, 40-fold, 50-fold, or 100-fold, in comparison to acondition in which a DNA-PK inhibitor and a genome editing system is notadministered to a cell(s), or compared to a condition in which only agenome editing system and not a DNA-PK inhibitor is administered to acell(s).

Use of DNA-PK Inhibitors and Genome Editing System, Kits, andCompositions Thereof

Genome editing, in which particular genomic regions are preciselyaltered, holds great therapeutic potential.

In some embodiments, provided herein are methods for editing one or moretarget genomic regions, for repairing a DNA break in one or more targetgenomic regions via a HDR pathway, for inhibiting or suppressingNHEJ-mediated repair of a DNA break in one or more target genomic, andfor modifying the expression of one or more genes or proteins viaadministering to a cell(s) a genome editing system and a DNA-PKinhibitor.

In some embodiments, provided herein are methods of modifying expressionof one or more genes or proteins comprising administering to one or morecells that comprise one or more target genomic regions, a genome editingsystem and a DNA-PK inhibitor described herein, wherein the genomeediting system interacts with a nucleic acid(s) of the one or moretarget genomic regions of a target gene(s), resulting in editing the oneor more target genomic regions and wherein the edit modifies expressionof a downstream gene (s) and/or protein(s) associated with the targetgene(s).

The genome editing system can be any genome editing system that can edita target genomic region in a cell(s). Exemplary genome editing systemsare described in detail above and can include, for example, ameganuclease based system, a zinc finger nuclease (ZFN) based system, aTranscription Activator-Like Effector-based Nuclease (TALEN) system, aCRISPR-based system, or NgAgo-based system

Editing of the one or more target genomic regions includes any kind ofgenetic manipulations or engineering of a cell's genome. The editing ofthe one or more target genomic regions can include insertions,deletions, or replacements of genomic regions in a cell(s) performed byone or more endonucleases. Genomic regions comprise the genetic materialin a cell(s), such as DNA, RNA, polynucleotides, and oligonucleotides.Genomic regions in a cell(s) also comprise the genomes of themitochondria or chloroplasts contained in a cell(s).

In some embodiments, provided herein are methods of treating a subjecthaving a disease or condition in need of editing one or more targetgenomic regions in a cell(s) of the subject, comprising administering toone or more cells a genomic editing system and a DNA-PK inhibitor.

In some embodiments, the methods provided herein are used to modifyexpression of a gene, an RNA molecule, a protein, a group of proteins,or downstream proteins in a pathway. Such modification can be used totreat a disease, a dysfunction, abnormal organismal homeostasis, eitheracquired or inherited or those due to the aging process. As used herein,the term “modify” or “modifying” includes modulating, enhancing,decreasing, increasing, inserting, deleting, knocking-out, knocking-in,and the like.

One of skill in the art understands that diseases, either acquired orinherited, or otherwise obtained, involve a dysregulation of homeostaticmechanisms including involvement of gene or protein function. To thisend, a skilled artisan can use the methods provided herein to modulate,modify, enhance, decrease, or provide an otherwise gene function in asubject.

Modifying expression of gene and consequent protein expression in acell(s) can be achieved by the methods provided herein, for example, byspecific editing (e.g., replacing, inserting or deleting, anycombinations thereof) a nucleic acid sequence in any of an exon, anintron, a transcription start site, a promoter region, an enhancerregion, a silencer region, an insulator region, an antirepressor, a posttranslational regulatory element, a polyadenylation signal (e.g. minimalpoly A), a conserved region, a transcription factor binding site, or anycombinations thereof.

In some embodiments, the methods, kits and compositions provided hereinare used to treat a subject that has cancer. The method of treating asubject having a cancer or cancer related condition comprisesadministering to a cell(s) of the subject a DNA-PK inhibitor and agenome editing system. The administration of the DNA-PK inhibitor andthe genome editing system can be in vivo or ex vivo.

The cancer can be of any kind of cancer. Cancer includes solid tumorssuch as breast, ovarian, prostate, lung, kidney, gastric, colon,testicular, head and neck, pancreas, brain, melanoma, and other tumorsof tissue organs and cancers of the blood cells, such as lymphomas andleukemias, including acute myelogenous leukemia, chronic lymphocyticleukemia, T cell lymphocytic leukemia, and B cell lymphomas. The cancerscan include melanoma, leukemia, astocytoma, glioblastoma, lymphoma,glioma, Hodgkins lymphoma, chronic lymphocyte leukemia and cancer of thepancreas, breast, thyroid, ovary, uterus, testis, pituitary, kidney,stomach, esophagus and rectum.

In some embodiments, the methods, kits and compositions provided hereinare used to treat a subject having any one or more of the followingcancers: Acute lymphoblastic leukemia (ALL), Acute myeloid leukemia,Adrenocortical carcinoma, AIDS-related cancers, AIDS-related lymphoma,Anal cancer, Appendix cancer, Astrocytoma, childhood cerebellar orcerebral, Basal-cell carcinoma, Bile duct cancer, extrahepatic (seecholangiocarcinoma), Bladder cancer, Bone tumor, osteosarcoma/malignantfibrous histiocytoma, Brainstem glioma, Brain cancer, Brain tumor,cerebellar astrocytoma, Brain tumor, cerebral astrocytoma/malignantglioma, Brain tumor, ependymoma, Brain tumor, medulloblastoma, Braintumor, supratentorial primitive neuroectodermal tumors, Brain tumor,visual pathway and hypothalamic glioma, Breast cancer, Bronchialadenomas/carcinoids, Burkitt's lymphoma, Carcinoid tumor, childhood,Carcinoid tumor, gastrointestinal, Carcinoma of unknown primary, Centralnervous system lymphoma, primary, Cerebellar astrocytoma, childhood,Cerebral astrocytoma/malignant glioma, childhood, Cervical cancer,Childhood cancers, Chondrosarcoma, Chronic lymphocytic leukemia, Chronicmyelogenous leukemia, Chronic myeloproliferative disorders, Coloncancer, Cutaneous T-cell lymphoma, Desmoplastic small round cell tumor,Endometrial cancer, Ependymoma, Epitheliod Hemangioendothelioma (EHE),Esophageal cancer, Ewing's sarcoma in the Ewing family of tumors,Extracranial germ cell tumor, Extragonadal germ cell tumor, Extrahepaticbile duct cancer, Eye cancer, intraocular melanoma, Eye cancer,retinoblastoma, Gallbladder cancer, Gastric (stomach) cancer,Gastrointestinal carcinoid tumor, Gastrointestinal stromal tumor (GIST),Germ cell tumor: extracranial, extragonadal, or ovarian, Gestationaltrophoblastic tumor, Glioma of the brain stem, Glioma, childhoodcerebral astrocytoma, Glioma, childhood visual pathway and hypothalamic,Gastric carcinoid, Hairy cell leukemia, Head and neck cancer, Heartcancer, Hepatocellular (liver) cancer, Hodgkin lymphoma, Hypopharyngealcancer, Hypothalamic and visual pathway glioma, childhood, Intraocularmelanoma, Islet cell carcinoma (endocrine pancreas), Kaposi sarcoma,Kidney cancer (renal cell cancer), Laryngeal cancer, Leukaemias,Leukaemia, acute lymphoblastic (also called acute lymphocyticleukaemia), Leukaemia, acute myeloid (also called acute myelogenousleukemia), Leukaemia, chronic lymphocytic (also called chroniclymphocytic leukemia), Leukemia, chronic myelogenous (also calledchronic myeloid leukemia), Leukemia, hairy cell, Lip and oral cavitycancer, Liposarcoma, Liver cancer (primary), Lung cancer, non-smallcell, Lung cancer, small cell, Lymphomas, AIDS-related Lymphoma, BurkittLymphoma, cutaneous T-Cell Lymphoma, Hodgkin Lymphomas, Non-Hodgkin (anold classification of all lymphomas except Hodgkin's) Lymphoma, primarycentral nervous system Macroglobulinemia, Waldenström, Male breastcancer, Malignant fibrous histiocytoma of bone/osteosarcoma,Medulloblastoma, childhood Melanoma, Melanoma, intraocular (eye), Merkelcell cancer, Mesothelioma, adult malignant Mesothelioma, childhoodMetastatic squamous neck cancer with occult primary, Mouth cancer,Multiple endocrine neoplasia syndrome Multiple myeloma/plasma cellneoplasm, Mycosis fungoides, Myelodysplastic syndromes,Myelodysplastic/myeloproliferative diseases, Myelogenous leukemia,chronic Myeloid leukemia, adult acute Myeloid leukemia, childhood acuteMyeloma, multiple (cancer of the bone-marrow), Myeloproliferativedisorders, chronic Myxoma, Nasal cavity and paranasal sinus cancer,Nasopharyngeal carcinoma, Neuroblastoma, Non-Hodgkin lymphoma, Non-smallcell lung cancer, Oligodendroglioma, Oral cancer, Oropharyngeal cancer,Osteosarcoma/malignant fibrous histiocytoma of bone, Ovarian cancer,Ovarian epithelial cancer (surface epithelial-stromal tumor), Ovariangerm cell tumor, Ovarian low malignant potential tumor, Pancreaticcancer, islet cell Pancreatic cancer, Paranasal sinus and nasal cavitycancer, Parathyroid cancer, Penile cancer, Pharyngeal cancer,Pheochromocytoma, Pineal astrocytoma, Pineal germinoma, Pineoblastomaand supratentorial primitive neuroectodermal tumors, Pituitary adenoma,Plasma cell neoplasia/Multiple myeloma, Pleuropulmonary blastoma,Primary central nervous system lymphoma, Prostate cancer, Rectal cancer,Renal cell carcinoma (kidney cancer), Renal pelvis and ureter caner,transitional cell cancer, Retinoblastoma, Rhabdomyosarcoma, Salivarygland cancer, Sarcoma, Ewing family of tumors, Kaposi Sarcoma, softtissue Sarcoma, uterine sarcoma, Sézary syndrome, Skin cancer(non-melanoma), Skin cancer (melanoma), Skin carcinoma, Merkel cell,Small cell lung cancer, Small intestine cancer, Soft tissue sarcoma,Squamous cell carcinoma—see skin cancer (non-melanoma), Squamous neckcancer with occult primary, metastatic, Stomach cancer, Supratentorialprimitive neuroectodermal tumor, T-Cell lymphoma, cutaneous (MycosisFungoides and Sézary syndrome), Testicular cancer, Throat cancer,Thymoma, Thymoma and thymic carcinoma, Thyroid cancer, Thyroid cancer,Transitional cell cancer of the renal pelvis and ureter, GestationalTrophoblastic tumor, Unknown primary site carcinoma of adult, Unknownprimary site cancer of, childhood, Ureter and renal pelvis, transitionalcell cancer, Urethral cancer, Uterine cancer, endometrial cancer,Uterine sarcoma, Vaginal cancer, Visual pathway and hypothalamic glioma,Vulvar cancer, Waldenström macroglobulinemia, or Wilms tumor (kidneycancer).

In some embodiments, exemplary target genes associated with cancerinclude ABL1, ABL2, ACSL3, AF15Q14, AF1Q, AF3p21, AF5q31, AKAP9, A T1,AKT2, ALDH2, AL, AL017, APC, ARHGEF12, ARHH, ARID1A, ARID2, ARNT,ASPSCR1, ASXL1, ATF1, ATIC, ATM, ATRX, AXIN1, BAP1, BCL10, BCL11A,BCL11B, BCL2, BCL3, BCLS, BCL6, BCL7A, BCL9, BCOR, BCR, BHD, BIRC3, BLM,BMPRIA, BRAF, BRCA1, BRCA2, BRD3, BRD4, BRIPI, BTG1, BUB1B, Cl2orf9,C15orf21, C15orf55, C16orf75, C2orf44, CAMTA1, CANT1, CARD11, CARS,CBFA2T1, CBFA2T3, C.BFB, CBL, CBLB, CBLC, CCDC6, CCNB1IP1, CCND1, CCND2,CCND3, CCNE1, CD273, CD274, CD74, CD79A, CD79B, CDH1, CDH11, CDK12,CDK4, CDK6, CD N2A, CD N2a(p14), CD N2C, CDX2, CEBPA, CEP1, CHCHD7,CHEK2, CHIC2, CHN1, CIC, Cin A, CLTC, CLTCL1, CMKOR1, CNOT3, COL1 A1,COPEB, COX6C, CREB1, CREB3 L1, CREB3 L2, CREBBP, CRLF2, CRTC3, CTNNB1,CYLD, D10S170, DAXX, DDB2, DDIT3, DDX10, DDX5, DDX6, DEK, D1CER1, DNM2,DNMT3A, DUX4, EBFI, ECT2 L, EGFR, E1F4A2, ELF4, ELK4, ELKS, ELL, ELN,EML4, EP300, EPS 15, ERBB2, ERCC2, ERCC3, ERCC4, ERCC5, ERG, ETV1, ETV4,ETV5, ETV6, EVI1, EWSR1, EXT1, EXT2, EZH2, EZR, FACL6, FAM22A, FAM22B,FAM46C, 1ANCA, EANCC, FANCD2, FANCE, FANCF, FANCG, FBXO1 1, FBXW7,FCGR2B, FEV, FGFR1, FGFRIOP, FGFR2, FGFR3, FTI, FIIIT, FIP1 L1, FLU,FLJ27352, FLT3, FNBP1, FOXL2, FOXOIA, FOX03A, FOXP1, FSTL3, FUBP1, FUS,FVT1, GAS7, GATA1, GATA2, GATA3, GMPS, GNA11, GNAQ, GNAS, GOLGAS, GOPC,GPC3, GPHN, GRAF, H3F3A, IICMOGT-1, IIEAB, HERPUD1, IIEY1, IIIP1,HIST1IT3B, IIIST1II4I, IILF, HLXB9, HMGA1, HMGA2, HNRNPA2BI, HOOK3,HOXA11, HOXA13, HOXA9, HOXC11, HOXC13, HOXD11, HOXD13, HRAS, IIRPT2,HSPCA, HSPCB, IDH1, IDH2, IGH, IGK, IGL, IKZF1, IL2, TL21R, IL6ST, IL7R,IRF4, IRTA1, ITK, JAK1, JAK2, JAK3, JAZF1, JUN, KCNJ5, KDM5A, KDM5C,KDM6A, KDR, KIAA1549, KIF5B, KIT, KLF4, KLK2, KRAS, KTN1, LAF4, LASP1,LCK, LCP1, LCX, LHFP, LIFR, LMO1, LM02, LPP, LRIG3, LYL1, MADH4, MAF,MAFB, MALT1, MAML2, MAP2KL MAP2K2, MλP2K4, MAX, MDM2, MDM4, MDS1, MDS2,MECT1, MED12, MEN1, MET, MITF, MKL1, MLF1, MLII1, MLL, MLL2, MLL3,MLLT1, MLLT10, MLLT2, MLLT3, MLLT4, MLLT6, MLLT7, MN1, MPL, MSF, MSH2,MSH6, MSI2, MSN, MTCP1, MUC1, MUTYH, MYB, MYC, MYCL1, MYCN, MYD88,MYH11, MYH9, MYST4, NACA, NBS1, NCOA1, NCOA2, NCOA4, NDRG1, NF1, NF2,NFE2 L2, NFIB, NFKB2, NIN, NKX2-1, NONO, NOTCH I, NOTCH2, NPM1, NR4A3,NRAS, NSD1, NT5C2, NTRK1, NTRK3, NUMA1, NUP214, NUP98, OLIG2, OMD,P2RY8, PAFAH1B2, PALB 2, PAX3, PAX5, PAX7, PAX8, PBRM1, PBX1, PCM1,PCSK7, PDE4DIP, PDGFB, PDGFRA, PDGFRB, PERI, PIIF6, PHOX2B, PICALM,PIK3CA, PIK3R1, PIM1, PLAG 1, PML, PMS1, PMS2, PMX1, PNUTL1, POT1,POU2AF1, POU5F1, PPARG, PPP2R1A, PRCC, PRDM1, PRDM16, PRF1, PRKAR1 A,PRO1073, PSIP2, PTCH, PTEN, PTPN11, RAB5EP, RAC1, RAD51 L1, RAF1,RALGDS, RANBP17, RAPIGDSI, RARA, RBI, RBM15, RECQL4, REL, RET, RNF43,ROS1, RPL10, RPL22, RPL5, RPN1, RUNDC2A, RUNX1, RUNXBP2, SBDS, SDC4,SDH5, SDHB, SDHC, SDHD, SEPT6, SET, SETBP1, SETD2, SF3B1, SFPQ, SFRS3,SH2B3, SH3GL1, SIL, SLC34A2, SLC45A3, SMARCA4, SMARCB1, SMARCE1, SMO,SOCS1, SOX2, SRGAP3, SRSF2, SSI8, SS18 L1, SSH3BP1, SSX1, SSX2, SSX4,STAT3, STK11, STL, SUFU, SIJZ12, SYK, TAF15, TALI, TAL2, TCEA1, TCF1,TCF12, TCF3, TCF7 L2, TCL1A, TCL6, TERT, TET2, TFE3, TFEB, TFG, TFPT,TFRC, THRAP3, TIF1, TLX1, TLX 3, TMPRSS2, TNFAIP3, TNFRSF14, TNFRSF17,TNFRSF6, TOPI, TP53, TPM3, TPM4, TPR, TRA, TRAF7, TRB, TRD, TRIM27,TRIM33, TRIP11, TSC1, TSC2, TSHR, TTL, U2AF1, USP6, VHL, VTUA, WAS,WHSC1, WHSC1 L1, WIF1, WRN, WT1, WTX, WWTR1, XPA, XPC, XPO1, YWHAE,ZNF145, ZNF198, ZNF278, ZNF331, ZNF384, ZNF521, ZNF9, ZRSR2 or anycombinations thereof.

In some embodiments, the methods provided herein are used to treat asubject that has an inherited disorder. The method of treating a subjecthaving a genetic disease or condition or inherited disorder, comprisesadministering to a cell(s) of the subject a DNA-PK inhibitor and agenome editing system. The administration of or the DNA-PK inhibitor andthe genome editing system can be in vivo or ex vivo.

The inherited disorder can result from mutations or duplications inchromosomal regions (e.g. from point mutations, deletions, insertions,frameshift, chromosomal duplications or deletions). The inheriteddisorder can be any inherited disorder.

In some embodiments, the inherited disorder is 22q11.2 deletionsyndrome, Angelman syndrome, Canavan disease, Charcot-Marie-Toothdisease, Color blindness, Cri du chat, Down syndrome, Duchenne musculardystrophy, Haemochromatosis, Haemophilia, Klinefelter syndrome,Neurofibromatosis, Phenylketonuria, Polycystic kidney disease,Prader-Willi syndrome, Sickle-cell disease, Spinal muscular atrophy,Spinal muscular atrophy, Tay-Sachs disease, Turner syndrome, ahemoglobinopathy, or any combinations thereof.

In some embodiments, the inherited disorder is 1p36 deletion syndrome,18p deletion syndrome, 21-hydroxylase deficiency, 47 XXX (triple Xsyndrome), 47 XXY (Klinefelter syndrome), 5-ALA dehydratase-deficientporphyria, ALA dehydratase deficiency, 5-aminolaevulinic dehydratasedeficiency porphyria, 5p deletion syndrome, Cri du chat (AKA5p-syndrome), ataxia telangiectasia (AKA A-T), alpha 1-antitrypsindeficiency (AAT), aceruloplasminemia, achondrogenesis type II (ACG2),achondroplasia (ACH), Acid beta-glucosidase deficiency, Gaucher disease(any type, e.g. type 1, type 2, type 3), Acrocephalosyndactyly (Apert),Apert syndrome, acrocephalosyndactyly (any type, e.g., type 1, type 2,type 3, type 5), Pfeiffer syndrome, Acrocephaly, Acute cerebralGaucher's disease, acute intermittent porphyria, (AIP) ACY2 deficiency,Alzheimer's disease (AD), Adelaide-type craniosynostosis, Muenkesyndrome, Adenomatous Polyposis Coli, familial adenomatous polyposis,Adenomatous Polyposis of the Colon, familial adenomatous polyposis(ADP), adenylosuccinate lyase deficiency, Adrenal gland disorders,Adrenogenital syndrome, Adrenoleukodystrophy, androgen insensitivitysyndrome (AIS), alkaptonuria (AKU), ALA dehydratase porphyria, ALA-Dporphyria, ALA dehydratase deficiency, Alagille syndrome, Albinism,Alcaptonuria, alkaptonuria, Alexander disease, alkaptonuria,Alkaptonuric ochronosis, alkaptonuria, alpha-1 proteinase inhibitordisease, alpha-1 related emphysema, Alpha-galactosidase A deficiency,Fabry disease, Alström syndrome, Alexander disease (ALX), Amelogenesisimperfecta, Amino levulinic acid dehydratase deficiency, Aminoacylase 2deficiency, Canavan disease, Anderson-Fabry disease, androgeninsensitivity syndrome, Anemia, hereditary sideroblastic, X-linkedsideroblastic anemiasplenic and/or familial anemia, AngiokeratomaCorporis Diffusum, Angiokeratoma diffuse, Angiomatosis retinae, vonHippel-Lindau disease, APC resistance, Leiden type, factor V Leidenthrombophilia, Apert syndrome, AR deficiency, androgen insensitivitysyndrome, Charcot-Marie-Tooth disease (any type, e.g., CMT1, CMTX, CMT2,CMT4, severe early onset CMT), Arachnodactyly, Marfan syndrome, ARNSHL,Nonsyndromic deafness (autosomal recessive, autosomal dominant,x-linked, or mitochondria), Arthro-ophthalmopathy, hereditaryprogressive, Stickler syndrome (e.g. COL2A1, COL11A1, COL11A2, COL9A1),Arthrochalasis multiplex congenita, Ehlers-Danlos syndrome (e.g.hypermobility type, arthrochalasia type, classical type, vascular type,kyphoscoliosis type, dermatosparaxis type) Asp deficiency, Aspadeficiency, Aspartoacylase deficiency, ataxia telangiectasia,Autism-Dementia-Ataxia-Loss of Purposeful Hand Use syndrome, Rettsyndrome, autosomal dominant juvenile ALS, Autosomal dominant opitzG/BBB syndrome, autosomal recessive form of juvenile ALS type 3,Amyotrophic lateral sclerosis (any type; e.g. ALS1, ALS2, ALS3, ALS4,ALS5, ALS5, ALS6, ALS7, ALS8, ALS9, ALS10, ALS11, ALS12, ALS13, ALS14,ALS15, ALS16, ALS17, ALS18, ALS19, ALS20, ALS21, ALS22, FTDALS1,FTDALS2, FTDALS3, FTDALS4, FTDALS4, IBMPFD2), Autosomal recessivenonsyndromic hearing loss, Autosomal Recessive Sensorineural HearingImpairment and Goiter, Pendred syndrome, Alexander disease (AxD), Ayerzasyndrome, famililal pulmonary arterial hypertension, B variant of theHexosaminidase GM2 gangliosidosis, Sandhoff disease, BANF-relateddisorder, neurofibromatosis (any type, e.g., NF1, NF2, schwannomatosis),Beare-Stevenson cutis gyrata syndrome, Benign paroxysmal peritonitis,Benjamin syndrome, beta-thalassemia, BH4 Deficiency, tetrahydrobiopterindeficiency, Bilateral Acoustic Neurofibromatosis, biotinidasedeficiency, bladder cancer, Bleeding disorders, factor V Leidenthrombophilia, Bloch-Sulzberger syndrome, incontinentia pigmenti, Bloomsyndrome, Bone diseases, Bourneville disease, tuberous sclerosis, Braindiseases, prion disease, breast cancer, Birt-Hogg-Dubé syndrome, Brittlebone disease, osteogenesis imperfecta, Broad Thumb-Hallux syndrome,Rubinstein-Taybi syndrome, Bronze Diabetes, hemochromatosis, Bronzedcirrhosis, Bulbospinal muscular atrophy, X-linked Spinal and bulbarmuscular atrophy, Burger-Grutz syndrome, lipoprotein lipase deficiency,familial CADASIL syndrome, CGD Chronic granulomatous disorder,Campomelic dysplasia, Cancer Family syndrome, hereditary nonpolyposiscolorectal cancer, breast cancer, bladder cancer, CarboxylaseDeficiency, Multiple Late-Onset biotinidase deficiency, Cat crysyndrome, Caylor cardiofacial syndrome, Ceramide trihexosidasedeficiency, Cerebelloretinal Angiomatosis, familial von Hippel-Lindaudisease, Cerebral arteriopathy, CADASIL syndrome, Cerebral autosomaldominant ateriopathy, CADASIL syndrome, Cerebroatrophic Hyperammonemia,Rett syndrome, Cerebroside Lipidosis syndrome, Charcot disease, CHARGEsyndrome, Chondrodystrophia, Chondrodystrophy syndrome, Chondrodystrophywith sensorineural deafness, otospondylomegaepiphyseal dysplasia,Chondrogenesis imperfecta, Choreoathetosis self-mutilation hyperuricemiasyndrome, Lesch-Nyhan syndrome, Classic Galactosemia, galactosemia,Cleft lip and palate, Stickler syndrome, Cloverleaf skull withthanatophoric dwarfism, Thanatophoric dysplasia (e.g. type 1 or type 2),Coffin-Lowry syndrome (CLS), Cockayne syndrome, Coffin-Lowry syndrome,collagenopathy types II and XI, familial Nonpolyposis, hereditarynonpolyposis colorectal cancer, familial Colon cancer, familialadenomatous polyposis, Colorectal cancer, Complete HPRT deficiency,Lesch-Nyhan syndrome, Complete hypoxanthine-guaninephosphoribosyltransferase deficiency, Compression neuropathy, hereditaryneuropathy with liability to pressure palsies, Connective tissuedisease, Conotruncal anomaly face syndrome, Cooley's Anemia,beta-thalassemia, Copper storage disease, Wilson's disease, Coppertransport disease, Menkes disease, Coproporphyria, hereditarycoproporphyria, Coproporphyrinogen oxidase deficiency, Cowden syndrome,CPX deficiency, Craniofacial dysarthrosis, Crouzon syndrome,Craniofacial Dysostosis, Crouzon syndrome, Crohn's disease,fibrostenosing, Crouzon syndrome, Crouzon syndrome with acanthosisnigricans, Crouzonodermoskeletal syndrome, Crouzonodermoskeletalsyndrome, Cockayne syndrome (CS), Cowden syndrome,Curschmann-Batten-Steinert syndrome, cutis gyrata syndrome ofBeare-Stevenson, Beare-Stevenson cutis gyrata syndrome, D-glyceratedehydrogenase deficiency, hyperoxaluria, primary, Dappled metaphysissyndrome, spondyloepimetaphyseal dysplasia, Strudwick type, DementiaAlzheimer's type (DAT), Genetic hypercalciuria, Dent's disease, musculardystrophy (e.g. Duchenne and Becker types), Deafness with goiter,Pendred syndrome, Deafness-retinitis pigmentosa syndrome, Ushersyndrome, Deficiency disease, Phenylalanine Hydroxylase, Degenerativenerve diseases, de Grouchy syndrome 1, De Grouchy syndrome,Dejerine-Sottas syndrome, Delta-aminolevulinate dehydratase deficiencyporphyria, Dementia, CADASIL syndrome, demyelinogenic leukodystrophy,Alexander disease, Dermatosparactic type of Ehlers-Danlos syndrome,Dermatosparaxis, inherited developmental disabilities, distal hereditarymotor neuropathy (dHMN), distal hereditary motor neuropathy (e.g.DHMN-V), DHTR deficiency, androgen insensitivity syndrome, DiffuseGloboid Body Sclerosis, Krabbe disease, Di George's syndrome,Dihydrotestosterone receptor deficiency, androgen insensitivitysyndrome, distal hereditary motor neuropathy, Myotonic dystrophy(type 1or type 2), distal spinal muscular atrophy (any type, including e.g.type 1, type 2, type 3, type 4, type 5, type 6), Duchenne/Beckermuscular dystrophy, Dwarfism (any kind, e.g.achondroplastic,achondroplasia, thanatophoric dysplasia), Dwarfism-retinalatrophy-deafness syndrome, Cockayne syndrome, dysmyelinogenicleukodystrophy, Alexander disease, Dystrophia myotonica, dystrophiaretinae pigmentosa-dysostosis syndrome, Usher syndrome, Early-Onsetfamilial alzheimer disease (EOFAD), Alzheimer disease (including e.g.type 1, type 2, type 3, or type 4) Ekman-Lobstein disease, osteogenesisimperfecta, Entrapment neuropathy, hereditary neuropathy with liabilityto pressure palsies, erythropoietic protoporphyria (EPP), Erythroblasticanemia, beta-thalassemia, Erythrohepatic protoporphyria, Erythroid5-aminolevulinate synthetase deficiency, X-linked sideroblastic anemia,Eye cancer, retinoblastoma FA—Friedreich ataxia, Friedreich's ataxia,FA, fanconi anemia, Facial injuries and disorders, factor V Leidenthrombophilia, FALS, amyotrophic lateral sclerosis, familial acousticneuroma, familial adenomatous polyposis, familial Alzheimer disease(FAD), familial amyotrophic lateral sclerosis, amyotrophic lateralsclerosis, familial dysautonomia, familial fat-inducedhypertriglyceridemia, lipoprotein lipase deficiency, familial, familialhemochromatosis, hemochromatosis, familial LPL deficiency, lipoproteinlipase deficiency, familial, familial nonpolyposis colon cancer,hereditary nonpolyposis colorectal cancer, familial paroxysmalpolyserositis, familial PCT, porphyria cutanea tarda, familialpressure-sensitive neuropathy, hereditary neuropathy with liability topressure palsies, familial primary pulmonary hypertension (FPPH),familial vascular leukoencephalopathy, CADASIL syndrome, FAP, familialadenomatous polyposis, FD, familial dysautonomia, Ferrochelatasedeficiency, ferroportin disease, Haemochromatosis (any type, e.g., type1, type 2A, type 2B, type 3, type 4, neonatal haemochromatosis,acaeruloplasminaemia, congenital atransferrinaemia, gracile syndrome)Periodic fever syndome, Familial Mediterranean fever (FMF), FG syndrome,FGFR3-associated coronal synostosis, Fibrinoid degeneration ofastrocytes, Alexander disease, Fibrocystic disease of the pancreas,Folling disease, fra(X) syndrome, fragile X syndrome, Fragilitas ossium,osteogenesis imperfecta, FRAXA syndrome, Friedreich's ataxia (FRDA),G6PD deficiency, Galactokinase deficiency disease, galactosemia,Galactose-1-phosphate uridyl-transferase deficiency disease,galactosemia, Galactosylceramidase deficiency disease, Krabbe disease,Galactosylceramide lipidosis, Krabbe disease, galactosylcerebrosidasedeficiency, galactosylsphingosine lipidosis, GALC deficiency, GALTdeficiency, galactosemia, Gaucher-like disease, pseudo-Gaucher disease,GBA deficiency, Genetic brain disorders, genetic emphysema, genetichemochromatosis, hemochromatosis, Giant cell hepatitis, neonatal,Neonatal hemochromatosis, GLA deficiency, Glioblastoma, retinal,retinoblastoma, Glioma, retinal, retinoblastoma, globoid cellleukodystrophy (GCL, GLD), Krabbe disease, globoid cellleukoencephalopathy, Glucocerebrosidase deficiency, Glucocerebrosidosis,Glucosyl cerebroside lipidosis, Glucosylceramidase deficiency,Glucosylceramide beta-glucosidase deficiency, Glucosylceramidelipidosis, Glyceric aciduria, hyperoxaluria, primary, Glycineencephalopathy, Nonketotic hyperglycinemia, Glycolic aciduria,hyperoxaluria, primary, GM2 gangliosidosis, Tay-Sachs disease,Goiter-deafness syndrome, Pendred syndrome, Graefe-Usher syndrome, Ushersyndrome, Gronblad-Strandberg syndrome, pseudoxanthoma elasticum,Haemochromatosis, hemochromatosis, Hallgren syndrome, Usher syndrome,Harlequin type ichthyosis, Hb S disease, hypochondroplasia(HCH),hereditary coproporphyria (HCP), Head and brain malformations, Hearingdisorders and deafness, Hearing problems in children, HEF2A, HEF2B,Hematoporphyria, porphyria, Heme synthetase deficiency, Hemochromatoses,hemoglobin M disease, methemoglobinemia beta-globin type, Hemoglobin Sdisease, hemophilia, hepatoerythropoietic porphyria (HEP), hepatic AGTdeficiency, hyperoxaluria, primary, Hepatolenticular degenerationsyndrome, Wilson disease, Hereditary arthro-ophthalmopathy, Sticklersyndrome, Hereditary dystopic lipidosis, Hereditary hemochromatosis(HHC), hemochromatosis, Hereditary hemorrhagic telangiectasia (HHT),Hereditary Inclusion Body Myopathy, skeletal muscle regeneration,Hereditary iron-loading anemia, X-linked sideroblastic anemia,Hereditary motor and sensory neuropathy, Hereditary motor neuronopathy,type V, distal hereditary motor neuropathy, Hereditary multipleexostoses, Hereditary nonpolyposis colorectal cancer, Hereditaryperiodic fever syndrome, Hereditary Polyposis Coli, familial adenomatouspolyposis, Hereditary pulmonary emphysema, Hereditary resistance toactivated protein C, factor V Leiden thrombophilia, Hereditary sensoryand autonomic neuropathy type III, familial dysautonomia, Hereditaryspastic paraplegia, infantile-onset ascending hereditary spasticparalysis, Hereditary spinal ataxia, Friedreich's ataxia, Hereditaryspinal sclerosis, Friedreich's ataxia, Herrick's anemia, HeterozygousOSMED, Weissenbacher-Zweymüller syndrome, Heterozygousotospondylomegaepiphyseal dysplasia, Weissenbacher-Zweymüller syndrome,HexA deficiency, Tay-Sachs disease, Hexosaminidase A deficiency,Tay-Sachs disease, Hexosaminidase alpha-subunit deficiency (any variant,e.g. variant A, variant B), Tay-Sachs disease, HFE-associatedhemochromatosis, hemochromatosis, HGPS, Progeria, Hippel-Lindau disease,von Hippel-Lindau disease, hemochromatosis (HLAH), distal hereditarymotor neuropathy (HMN V), hereditary nonpolyposis colorectal cancer(HNPCC), hereditary neuropathy with liability to pressure palsies(HNPP), homocystinuria, Homogentisic acid oxidase deficiency,alkaptonuria, Homogentisic acidura, alkaptonuria, Homozygous porphyriacutanea tarda, hepatoerythropoietic porphyria, hyperoxaluria, primary(HP1), hyperoxaluria (HP2), hyperphenylalaninemia (HPA),HPRT—Hypoxanthine-guanine phosphoribosyltransferase deficiency,Lesch-Nyhan syndrome, HSAN type III, familial dysautonomia, familialdysautonomia (HSAN3), Hereditary Sensory Neuropathy (any type, e.g.HSN-1, HSN-II, HSN-III), familial dysautonomia, Human dermatosparaxis,Huntington's disease, Hutchinson-Gilford progeria syndrome, progeria,Hyperandrogenism, nonclassic type due to 21-hydroxylase deficiency,Hyperchylomicronemia, familial lipoprotein lipase deficiency, familial,Hyperglycinemia with ketoacidosis and leukopenia, propionic acidemia,Hyperlipoproteinemia type I, lipoprotein lipase deficiency, familialhyperoxaluria, primary hyperphenylalaninaemia, hyperphenylalaninemia,hyperphenylalaninemia, Hypochondrodysplasia, hypochondroplasia,Hypochondrogenesis, Hypochondroplasia, Hypochromic anemia, X-linkedsideroblastic anemia, Hypoxanthine phosphoribosyltransferse (HPRT)deficiency, Lesch-Nyhan syndrome, , infantile-onset ascending hereditaryspastic paralysis (IAHSP), ICF syndrome, Immunodeficiency, centromereinstability and facial anomalies syndrome, Idiopathic hemochromatosis,hemochromatosis, type 3, Idiopathic neonatal hemochromatosis,hemochromatosis, neonatal, Idiopathic pulmonary hypertension, Immunesystem disorders, X-linked severe combined immunodeficiency,Incontinentia pigmenti, Infantile cerebral Gaucher's disease, InfantileGaucher disease, infantile-onset ascending hereditary spastic paralysis,Infertility, inherited emphysema, inherited tendency to pressurepalsies, hereditary neuropathy with liability to pressure palsies,Insley-Astley syndrome, otospondylomegaepiphyseal dysplasia,Intermittent acute porphyria syndrome, acute intermittent porphyria,Intestinal polyposis-cutaneous pigmentation syndrome, Peutz-Jegherssyndrome, incontinentia pigmenti (IP), Iron storage disorder,hemochromatosis, Isodicentric 15, isodicentric 15, Isolated deafness,nonsyndromic deafness, Jackson-Weiss syndrome, Joubert syndrome,Juvenile Primary Lateral Sclerosis (JPLS), juvenile amyotrophic lateralsclerosis, Juvenile gout, choreoathetosis, mental retardation syndrome,Lesch-Nyhan syndrome, juvenile hyperuricemia syndrome, Lesch-Nyhansyndrome, Jackson-Weiss syndrome (JWS), spinal and bulbar muscularatrophy, Kennedy disease, spinal and bulbar muscular atrophy, Kennedyspinal and bulbar muscular atrophy, spinal and bulbar muscular atrophy,Kerasin histiocytosis, Kerasin lipoidosis, Kerasin thesaurismosis,ketotic glycinemia, propionic acidemia, ketotic hyperglycinemia,propionic acidemia, Kidney diseases, hyperoxaluria, primary, Kniestdysplasia, Krabbe disease, Kugelberg-Welander disease, spinal muscularatrophy, Lacunar dementia, CADASIL syndrome, Langer-Saldinoachondrogenesis, Langer-Saldino dysplasia, Late-onset Alzheimer disease,late-onset Krabbe disease (LOKD), Krabbe disease, Learning Disorders,Learning disability, Lentiginosis, perioral, Peutz-Jeghers syndrome,Lesch-Nyhan syndrome, Leukodystrophies, leukodystrophy with Rosenthalfibers, Alexander disease, Leukodystrophy, spongiform, Li-Fraumenisyndrome (LFS), Li-Fraumeni syndrome, Lipase D deficiency, lipoproteinlipase deficiency, familial LIPD deficiency, lipoprotein lipasedeficiency, familial Lipidosis, cerebroside, Lipidosis, ganglioside,infantile, Tay-Sachs disease, Lipoid histiocytosis (kerasin type),lipoprotein lipase deficiency, familial Liver diseases, galactosemia,Lou Gehrig disease, Louis-Bar syndrome, ataxia telangiectasia, Lynchsyndrome, hereditary nonpolyposis colorectal cancer, Lysyl-hydroxylasedeficiency, Machado-Joseph disease, Spinocerebellar ataxia (any type,e.g. SCA1, SCA2, SCA3, SCA 18, SCA20, SCA21, SCA23, SCA26, SCA28,SCA29), Male breast cancer, breast cancer, Male genital disorders,Malignant neoplasm of breast, breast cancer, malignant tumor of breast,breast cancer, Malignant tumor of urinary bladder, bladder cancer,Mammary cancer, breast cancer, Marfan syndrome, Marker X syndrome,fragile X syndrome, Martin-Bell syndrome, fragile X syndrome,McCune-Albright syndrome, McLeod syndrome, MEDNIK syndrome,Mediterranean Anemia, beta-thalassemia, Mega-epiphyseal dwarfism,otospondylomegaepiphyseal dysplasia, Menkea syndrome, Menkes disease,Menkes disease, Mental retardation with osteocartilaginousabnormalities, Coffin-Lowry syndrome, Metabolic disorders, Metatropicdwarfism, type II, Kniest dysplasia, Metatropic dysplasia type II,Kniest dysplasia, Methemoglobinemia (any type, e.g. congenital,beta-globin type, congenital methemoglobinemia type II), methylmalonicacidemia, Marfan syndrome (MFS), MHAM, Cowden syndrome, Micro syndrome,Microcephaly, MMA, methylmalonic acidemia, Menkes disease (AKA MK orMNK), Monosomy 1p36 syndrome, Motor neuron disease, amyotrophic lateralsclerosis, amyotrophic lateral sclerosis, Movement disorders,Mowat-Wilson syndrome, Mucopolysaccharidosis (MPS I), Mucoviscidosis,Multi-Infarct dementia, CADASIL syndrome, Multiple carboxylasedeficiency, late-onset, biotinidase deficiency, Multiple hamartomasyndrome, Cowden syndrome, Multiple neurofibromatosis, Musculardystrophy (any type, including,e.g., Duchenne and Becker type), Myotoniaatrophica, myotonic dystrophy, Myotonia dystrophica, Nance-Insleysyndrome, otospondylomegaepiphyseal dysplasia, Nance-Sweeneychondrodysplasia, otospondylomegaepiphyseal dysplasia, NBIA1,pantothenate kinase-associated neurodegeneration, Neill-Dingwallsyndrome, Cockayne syndrome, Neuroblastoma, retinal, retinoblastoma,Neurodegeneration with brain iron accumulation type 1, pantothenatekinase-associated neurodegeneration, Neurologic diseases, Neuromusculardisorders, distal hereditary motor neuronopathy, Niemann-Pick,Niemann-Pick disease, Noack syndrome, Nonketotic hyperglycinemia,Glycine encephalopathy, Non-neuronopathic Gaucher disease,Non-phenylketonuric hyperphenylalaninemia, tetrahydrobiopterindeficiency, nonsyndromic deafness, Noonan syndrome, Norrbottnian Gaucherdisease, Ochronosis, alkaptonuria, Ochronotic arthritis, alkaptonuria,Ogden syndrome, osteogenesis imperfecta (OD, Osler-Weber-Rendu disease,Hereditary hemorrhagic telangiectasia, OSMED, otospondylomegaepiphysealdysplasia, osteogenesis imperfecta, Osteopsathyrosis, osteogenesisimperfecta, Osteosclerosis congenita, Oto-spondylo-megaepiphysealdysplasia, otospondylomegaepiphyseal dysplasia,otospondylomegaepiphyseal dysplasia, Oxalosis, hyperoxaluria, primary,Oxaluria, primary, hyperoxaluria, primary, pantothenatekinase-associated neurodegeneration, Patau Syndrome (Trisomy 13), PBGDdeficiency, acute intermittent porphyria, PCC deficiency, propionicacidemia, porphyria cutanea tarda (PCT), PDM disease, Pendred syndrome,Periodic disease, Mediterranean fever, Familial Periodic peritonitis,Periorificial lentiginosis syndrome, Peutz-Jeghers syndrome, Peripheralnerve disorders, familial dysautonomia, Peripheral neurofibromatosis,Peroneal muscular atrophy, peroxisomal alanine:glyoxylateaminotransferase deficiency, hyperoxaluria, primary Peutz-Jegherssyndrome, Phenylalanine hydroxylase deficiency disease,Pheochromocytoma, von Hippel-Lindau disease, Pierre Robin syndrome withfetal chondrodysplasia, Weissenbacher-Zweymüller syndrome, Pigmentarycirrhosis, hemochromatosis, Peutz-Jeghers syndrome (PJS), pantothenatekinase-associated neurodegeneration (PKAN), PKU, phenylketonuria,Plumboporphyria, ALA deficiency porphyria, PMA, Polycystic kidneydisease, polyostotic fibrous dysplasia, McCune-Albright syndrome,familial adenomatous polyposis, hamartomatous intestinal polyposis,polyps-and-spots syndrome, Peutz-Jeghers syndrome, Porphobilinogensynthase deficiency, ALA deficiency porphyria, porphyrin disorder, PPDXdeficiency, variegate porphyria, Prader-Labhart-Willi syndrome,Prader-Willi syndrome, presenile and senile dementia, Primary ciliarydyskinesia (PCD), primary hemochromatosis, hemochromatosis, primaryhyperuricemia syndrome, Lesch-Nyhan syndrome, primary seniledegenerative dementia, procollagen type EDS VII, mutant, progeria,Hutchinson Gilford Progeria Syndrome, Progeria-like syndrome, Cockaynesyndrome, progeroid nanism, Cockayne syndrome, progressive chorea,chronic hereditary (Huntington), Huntington's disease, progressivelydeforming osteogenesis imperfecta with normal sclerae, Osteogenesisimperfecta (any type, e.g. Type I, Type II, Type III, Type IV, Type V,Type VI, Type VII, Type VIII), proximal myotonic dystrophy (PROMM),propionic acidemia, propionyl-CoA carboxylase deficiency, protein Cdeficiency, protein S deficiency, protoporphyria, protoporphyrinogenoxidase deficiency, variegate porphyria, proximal myotonic dystrophy,Myotonic dystrophytype 2, proximal myotonic myopathy, pseudo-Gaucherdisease, pseudoxanthoma elasticum, psychosine lipidosis, Krabbe disease,pulmonary arterial hypertension, pulmonary hypertension, pseudoxanthomaelasticum (PXE), pseudoxanthoma elasticum, retinoblastoma (Rb),Recklinghausen disease, Recurrent polyserositis, Retinal disorders,Retinitis pigmentosa-deafness syndrome, Usher syndrome, Retinoblastoma,Rett syndrome, RFALS type 3, Ricker syndrome, Riley-Day syndrome,familial dysautonomia, Roussy-Levy syndrome, Rubinstein-Taybi syndrome(RSTS), Rett syndrome (RTS), Rubinstein-Taybi syndrome, Rubinstein-Taybisyndrome, Sack-Barabas syndrome, SADDAN disease, sarcoma family syndromeof Li and Fraumeni, Li-Fraumeni syndrome, SBLA syndrome (sarcoma,breast, leukemia, and adrenal gland syndrome), Li-Fraumeni syndrome,Spinal and bulbar muscular atrophy (SBMA), Schwannoma, acoustic,bilateral, neurofibromatosis type II, Schwartz-Jampel syndrome, X-linkedsevere combined immunodeficiency (SCIDX1), SED congenita,spondyloepiphyseal dysplasia congenita, SED Strudwick,spondyloepimetaphyseal dysplasia, Strudwick type, spondyloepiphysealdysplasia congenita (SEDc), Spondyloepimetaphyseal dysplasia (SEMD),Strudwick type SEMD, senile dementia, severe achondroplasia withdevelopmental delay and acanthosis nigricans, SADDAN disease, Shprintzensyndrome, Siderius X-linked mental retardation syndrome caused bymutations in the PHF8 gene, skeleton-skin-brain syndrome, Skinpigmentation disorders, spinal muscular atrophy (SMA),Spondylo-meta-epiphyseal dysplasia (SMED) (any type, e.g. Studwick type,type 1), Smith-Lemli-Opitz syndrome, Smith Magenis Syndrome,South-African genetic porphyria, infantile onset ascending spasticparalysis, infantile-onset ascending hereditary spastic paralysis,Speech and communication disorders, sphingolipidosis, Tay-Sachs,Tay-Sachs disease, spinal and bulbar muscular atrophy, spinal muscularatrophy, spinal muscular atrophy, distal type V, distal hereditary motorneuropathy, spinal muscular atrophy distal with upper limb predominance,distal hereditary motor neuropathy, spinocerebellar ataxia,spondyloepiphyseal dysplasia congenita, spondyloepiphyseal dysplasia,collagenopathy(any type, e.g. types II and XI), spondyloepimetaphysealdysplasia, spondylometaphyseal dysplasia (SMD), spondyloepimetaphysealdysplasia, spongy degeneration of central nervous system, spongydegeneration of the brain, spongy degeneration of white matter ininfancy, sporadic primary pulmonary hypertension, SSB syndrome, steelyhair syndrome, Menkes disease, Steinert disease, myotonic dystrophy,Steinert myotonic dystrophy syndrome, myotonic dystrophy, Sticklersyndrome, stroke, CADASIL syndrome, Strudwick syndrome, subacuteneuronopathic Gaucher disease, Swedish genetic porphyria, acuteintermittent porphyria, acute intermittent porphyria, Swiss cheesecartilage dysplasia, Kniest dysplasia, Tay-Sachs disease,TD—thanatophoric dwarfism, thanatophoric dysplasia, TD with straightfemurs and cloverleaf skull, thanatophoric dysplasia Type 2,Telangiectasia, cerebello-oculocutaneous, ataxia telangiectasia,Testicular feminization syndrome, androgen insensitivity syndrome,tetrahydrobiopterin deficiency, testicular feminization syndrome (TFM),androgen insensitivity syndrome, thalassemia intermedia,beta-thalassemia, Thalassemia Major, beta-thalassemia, thanatophoricdysplasia, Thrombophilia due to deficiency of cofactor for activatedprotein C, Leiden type, factor V Leiden thrombophilia, Thyroid disease,Tomaculous neuropathy, hereditary neuropathy with liability to pressurepalsies, Total HPRT deficiency, Lesch-Nyhan syndrome, Totalhypoxanthine-guanine phosphoribosyl transferase deficiency, Lesch-Nyhansyndrome, Treacher Collins syndrome, Trias fragilitis ossium, triple Xsyndrome, Triplo X syndrome, Trisomy 21Trisomy X,Troisier-Hanot-Chauffard syndrome, hemochromatosis, Tay-Sachs disease(TSD), Tuberous Sclerosis Complex (TSC), Tuberous sclerosis, Turner-likesyndrome, Noonan syndrome, UDP-galactose-4-epimerase deficiency disease,galactosemia, UDP glucose 4-epimerase deficiency disease, galactosemia,UDP glucose hexose-1-phosphate uridylyltransferase deficiency,galactosemia, Undifferentiated deafness, nonsyndromic deafness, UPSdeficiency, acute intermittent porphyria, Urinary bladder cancer,bladder cancer, UROD deficiency, Uroporphyrinogen decarboxylasedeficiency, Uroporphyrinogen synthase deficiency, acute intermittentporphyria, Usher syndrome, UTP hexose-1-phosphate uridylyltransferasedeficiency, galactosemia, Van Bogaert-Bertrand syndrome, Van der Hoevesyndrome, Velocardiofacial syndrome, VHL syndrome, von Hippel-Lindaudisease, Vision impairment and blindness, Alström syndrome, VonBogaert-Bertrand disease, von Hippel-Lindau disease, VonRecklenhausen-Applebaum disease, hemochromatosis, von Recklinghausendisease, neurofibromatosis type I, Vrolik disease, osteogenesisimperfecta, Waardenburg syndrome, Warburg Sjo Fledelius Syndrome, Microsyndrome, Wilson disease (WD), Weissenbacher-Zweymüller syndrome,Werdnig-Hoffmann disease, spinal muscular atrophy, Williams Syndrome,Wilson disease, Wilson's disease, Wilson disease, Wolf-Hirschhornsyndrome, Wolff Periodic disease, Weissenbacher-Zweymüller syndrome(WZS), Xeroderma pigmentosum, X-linked mental retardation andmacroorchidism, fragile X syndrome, X-linked primary hyperuricemia,Lesch-Nyhan syndrome, X-linked severe combined immunodeficiency,X-linked sideroblastic anemia, X-linked spinal-bulbar muscle atrophy,spinal and bulbar muscular atrophy, X-linked uric aciduria enzymedefect, Lesch-Nyhan syndrome, X-SCID, X-linked severe combinedimmunodeficiency, X-linked sideroblastic anemia (XLSA), X-SCID, X-linkedsevere combined immunodeficiency, X-linked sideroblastic anemia (XLSA),XSCID, X-linked severe combined immunodeficiency, XXX syndrome, triple Xsyndrome, XXXX syndrome, XXXXX syndrome, XXXXX, XXY syndrome, XXYtrisomy, Klinefelter syndrome, XYY syndrome, triplet repeat disorders,or any combinations thereof.

In embodiments, a specific post-transcriptional control modulator istargeted for modulation, modification, enhancement or decrease inactivity by administering a DNA-PK inhibitor and a genomic editingsystem. For example, post-transcriptional control modulators can includePARN, PAN, CPSF, CstF, PAP, PABP, PAB2, CFI, CFII, RNA triphosphatase,RNA gluanyltransferase, RNA methyltransferase, SAM synthase,ubiquitin-conjugating enzyme E2R, SR proteins SFRS1 through SFR11, hnRNPproteins (e.g. HNRNPAO, HNRNPA1, HNRNPA1 L1, HNRNPA1 L2, HNRNPA2,HNRNPA2B1, HNRNPAB, HNRNPB1, HNRNPC, HNRNPCL1, HNRNPD, HNRPDL, HNRNPF,HNRNHP1, HNRNPH2, HNRNPH3, HNRNPK, HNRNPL, HNRNPLL, HNRNPM, HNRNPR,HNRNPU, HNRNPUL1, HNRNPUL2, HNRNPUL3, ADAR, Mex 67, Mtr2, Nab2, Dead-boxhelicase, e1F4A, e1F4B, e1F4E, e1F4G, GEF, GCN2, PKR, HRI, PERK, eEF1,eEF2, GCN, eRF3, ARE-specific binding proteins, EXRN1, DCP1, DCP2,RCK/p54, CPEB, eIF4E, microRNAS and siRNAs, DICER, Ago proteins,Nonsence-mediated mRNA decay proteins, UPF3A, UPF3BeIF4A3, MLN51,Y14/MAGOH, MG-1, SMG-5, SMG-6, SMG-7, or any combinations thereof.

In some embodiments, genetic pathways associated with the cell cycle aremodulated, enhanced or decreased in activity by administering a DNA-PKinhibitor and a genomic editing system. Exemplary pathways and genesassociated with the cell cycle include ATM, PMS2, FAS-L, MRE11, MLH1,FasR, NBS1, MSH6, Trail-L, RAD50, MSH2, Trail-R, 53BP1, RFC, TNF-Ct,P53, PCNA, TNF-R1, CHKE, MSH3, FADD, E2F1, MutS, homolog, TRADD, PML,MutL, homolog, R1P1, FANCD2, Exonuclease, MyD88, SMC1, DNA, Polymerase,delta, IRAK, BLM1, (POLD1, POLD2, POLD3, NIL, BRCA1, and, POLD4, -genes,IKK, H2AX, encoding, subunits), NFKβ, ATR, Topoisomerase, 1, IκBα, RPA,Topoisomerase, 2, IAP, ATRIP, RNAseHl, Caspase, 3, RADS, Ligase, 1,Caspase, 6, RAD1, DNA, polymerase, 1, Caspase, 7, HUS, DNA, polymerase,3, Caspase, 8, RAD17, Primase, Caspase, 10, RFC, Helicase, HDAC1, CHK1,Single strand, binding, HDAC2, TLK1, proteins, Cytochrome, C, CDC25,Bx1-xL, STAT3, STAT5, DFF45, Vcl-2, ENDO-G, PI3K, Akt, Calpain, Bad,Bax, Ubiqiiitin-mediated proteolysis, Hypoxia, Cell Proliferation,HIF-loc, MAPK, E1, HERC1, TRAF6, HIF-Iβ, MAPKK, E2, UBE2Q, MEKK1, Ref1,MAPKKK, E3, UBE2R, COP!, HSP90, c-Met, UBLE1A, UBE2S, PIFH2, VEGF, HGF,UBLE1B, UBE2Q, cIAP, PAS, ER, S1/2, UBLEIC, UBE2W, PIAS, ARNT, ATK,UBE2A, UBE2Z, SYVN, VHL, PKCs, UBE2B, AFC, LLC, N, NHLRC1, HLF, Paxilin,UBE2C, UBE1, AIRE, EPF, FAK, UBE2A, E6AP, MGRN1, VDU2, Adducin, UBE2E,UBE3B, BRCA1, SUMORESUME, PYK1, UBE2F, Smurf, FANCL, SENP1, RB, UBE2G1,Itch, MIDI, Calcineurin, A, RBI, UBE2G2, HERC2, Cdc20, RACK1, Raf-1,UBE2I, HERC3, Cdhl, PTB, A-Raf, UBE2J1, HERC4, Apcl, Hur, B-raf, UBE2J2,UBE4A, Apc2, PHD2, MEK1/2, UBE2 L3, UBE4B, Apc3, SSAT2, ERK1/2, UBE2 L6,CHIP, Apc4, SSAT1, Ets, UBE2M, CYC4, Apc5, GSK3, Elkl, UBE2N, PPR19,Apc6, CBP, SAP1, UBE20, UIPS, Apc7, FOX04, cPLA2, WWPI, Mdm2, Apc8,F1H-1, WWP2, Parkin, Apc9, TRIP, 12, Trim32, Ape, 10, NEED4, Trim37,Ape, 11, ARF-BP1, SIAH-1, Ape, 12, EDD1, PML, Cell, survival, Cell,cycle, arrest, SMADI, P21, SMADS5, BAX, SAMD8, MDR, LEF1, DRAIL, IGFBP3,TCF3, GADD45, TCF4, P300, HAT1, PI3, Akt, GF1, or any combinationsthereof.

In some embodiments, genes associated with angiogenesis are modulated,enhanced or decreased in activity by administering a DNA-PK inhibitorand a genomic editing system to a cell(s). Exemplary genes and geneticpathways associated with angiogenesis, and angiogenesis-relatedconditions include VEGF, VEGFR2, SHC, E2F7, VEGFB, VEGFR3, PI3, VEGFC,Nrp 1, PIP3, EGFDIP3, DAG, GRB2, SOS, Akt, PB, PKC, Ras, RAF1, DAG,eNOS, NO, ERK1, ER2, cPLA2, ME1, MEK2, or any combinations thereof.

In some embodiments, genetic pathways and/or genes associated withmitochondrial function are modulated, enhanced or decreased in activityby administering a DNA-PK inhibitor and a genomic editing system to acell(s). Exemplary genes and genetic pathways associated withmitochondrial function include Malate dehydrogenase Aminotransferase,Hydratase, Deacylase, Dehydrogenase, Carboxylase, Mutase, Fatty acidoxidation Leucine Oxidation Isoleucine disorders (enzyme Pathwayoxidation pathway deficiencies) Aminotransferase Aminotransferase, OCTN2Branched chain Branched chain, FATP1-6 aminotransferase 2,aminotransferase 2, CPT-1 mitochondrial mitochondrial, CACTIsobutytyl-CoA 2-methylbutytyl-CoA, CPT-II dehydrogenase Dehydrogenase,SCAD (Branched Chain (Branched Chain, MCAD Keto Acid Keto Acid, VLCADDehydrogase Dehydrogenase, ETF-DH Complex) Complex), Alpha-ETF HydrataseHydratase, Beta-ETF HMG-CoA lyase 2-methyl-3-OH-SCHAD butyryl-CoA, LCHADdehydrogenase, MTP 3-Oxothiolase, LKAT,DECR 1, HMGCS2, HMGCL, or anycombinations thereof.

In some embodiments, genetic pathways and/or genes associated with DNAdamage or genomic instability are modulated, enhanced or decreased inactivity. Exemplary genes and genetic pathways associated with pathwaysand/or genes relating to DNA Damage and genomic instability include53BP1, BLM, MBD2, DNA, ligase, 4, MDC1, H2AX, XLF, SMC1, 53BP1, Rad50,P53, P53, Artemis, Rad27, TdT, APE1, PMS2, APE2, UvrA, RecA, MLH1,NEIL1, UvrB, SSB, MSH6, NEIL2, UvrC, Mrell, MSH2, NEIL3, XPC, Rad50,RFC, XRCC1, Rad23B, Nbsl, PCNA, PNKP, CEN2, CtIP, MSH3, Tdpl, DDB1, RPA,MutS, APTX, XPE, Rad51, MutL, DNA, polymerase β CSA, Rad52, DNApolymerase δ, CSB, Rad54, Topoisomerase, 1, DNA, TFT1H, BRCA1,Topoisomerase, 2, PCNA, XPB, BRCA2, RNAseHl, FEN1, XPD, Exol, Ligase 1,RFC, XPA, BLM, DNA, polymerase, 1, PAR, 1, RPA, Top111a, DNA, Lig1, XPG,GEN1, Primase, Lig3, ERCC1 Yen1 Helicase, UNG, XPF, Slxl, SSBs, MUTY DNApolymerase δ, Slx4, SMUG DNA polymerase ε, Mus8, MBD4, Emel, Dssl, ASH1L, SETD4, DQT1 L, SETDS, EHMT1, SETD6, EHMT2, SETD7, EZH1, SETD8, EZH2,SETD9, MLL, SETDB1, MLL2, SETDB2, MLL3, SETMAR, MLL4, SMYD, 1, MLLS,SMYD2, NSD, 1, SMYD3, PRDM2, SMYD4, SET, SMYD5, SETBP1, SUV39H1, SETD1A, SUV39H2, SETD 1B, SUV420H1, SETD2, SUV420 H2, SETD3, or anycombinations thereof.

In some embodiments, genes encoding for mammalian transcription factorsare modulated, enhanced, decreased or provided to a cell. Exemplaryhuman transcription factors include AFF4, AFF3, AFF2, AFF1, AR, TFAP2B,TFAP2D, TFAP2C, TFAP2E, TFAP2A, JARID2, KDM5D, ARID4A, ARID4B, KDM5A,ARID3A, KDM5B, KDM5C, ARID5B, ARID3B, ARID2, ARID5A, ARID3C, ARID1A,ARID1B, HIF1A, NPAS1, NPAS3, NPAS4, MLXIPL, ARNTL2, MXD1, AHRR, TFE3,HES2, MNT, TCF3, SREBF1, TFAP4, TCFL5, LYL1, USF2, TFEC, AHR, MLX, MYF6,MYF5, SIM1, TFEB, HAND1, HES1, ID2, MYCL1, ID3, TCF21, MXI1, SOHLH2,MYOG, TWIST1, NEUROG3, BHLHE41, NEUROD4, MXD4, BHLHE23, TCF15, MAX, ID1,MYOD1, ARNTL, BHLHE40, MYCN, CLOCK, HEY2, MYC, ASCL1, TCF12, ARNT, HES6,FERD3 L, MSGN1, USF1, TALI, NEUROD1, TCF23, HEYL, HAND2, NEUROD6, HEY1,SOHLH1, MESP1, PTF1A, ATOH8, NPAS2, NEUROD2, NHLH1, ID4, ATOH1, ARNT2,HES3, MLXIP, ASCL3, KIAA2018, OLIG3, NHLH2, NEUROG2, MSC, HES7, ATOH7,BHLHA15, BHLHE22, NEUROG1, FIGLA, ASCL2, OLIG1, TAL2, MITF, SCXB, HELT,ASCL4, MESP2, HES4, SCXA, TCF4, HES5, SREBF2, BHLHA9, OLIG2, MXD3,TWIST2, LOC388553, C13orf38-SOHLH2, CEBPE, XBP1, BATF3, CREB5, CEBPG,ATF3, ATF7, CEBPB, CEBPD, CEBPA, CBFB, CAMTA2, CAMTA1, EBF4, EBF3, EBF1,EBF2, NR2F6, NR2F1, NR2F2, GRHL2, TFCP2 L1, GRHL1, TFCP2, UBP1, GRHL3,YBX2, CSDE1, CSDA, YBX1, LIN28A, CARHSP1, CSDC2, LIN28B, NFIX, NFIC,NFIB, NFIA, CUX2, ONECUT2, CUX1, ONECUT1, SATB1, ONECUT3, SATB2, DMRT3,DMRT1, DMRTC2, DMRTA2, DMRTB1, DMRT2, DMRTA1, E2F2, E2F1, E2F3, TFDP2,E2F8, E2F5, E2F7, E2F6, TFDP3, TFDP1, E2F4, NR1H3, NR1H2, ETV1, ETV7,SPI1, ELF4, ETV2, ERF, ELF2, ELK3, ETV3, ELF1, SPDEF, ELK1, ETS1, EHF,ELF5, ETV6, SPIB, FLI1, GABPA, ERG, ETS2, ELK4, ELF3, FEY, SPIC, ETV4,ETV5, FOXN3, FOXC1, FOXJ2, FOXF1, FOXN1, FOXM1, FOXP1, FOXO3, FOXA2,FOXP2, FOXJ1, FOXP4, FOXF2, FOXN4, FOXK2, FOXO1, FOXH1, FOXQ1, FOXK1,FOXI1, FOXD4, FOXA3, FOXN2, FOXB1, FOXG1, FOXR1, FOXL1, FOXC2, FOXE1,FOXS1, FOXL2, FOXO4, FOXD4 L1, FOXD4 L4, FOXD2, FOXI2, FOXE3, FOXD3,FOXD4 L3, FOXR2, FOXJ3, FOXO6, FOXB2, FOXD4 L5, FOXD4 L6, FOXD4 L2,KIAA0415, FOXA1, FOXP3, GCM2, GCM1, NR3C1, GTF2IRD1, GTF2I, GTF2IRD2B,GTF2IRD2, SOX8, SOX30, PMS1, CIC, TCF7, TOX4, SOX10, HMGXB4, HBP1, TFAM,UBTF, WHSC1, SOX6, HMGXB3, BBX, TOX2, SOX4, SOX21, SOX9, SOX15, SOX5,SOX3, LEF1, HMG20A, SOX13, TCF7 L2, SSRP1, TCF7 L1, SOX17, SOX14, PINX1,SOX7, SOX11, SOX12, SOX2, SOX1, SRY, SOX18, UBTFL1, UBTFL2, TOX, HMGB1,HMGB2, PBRM1, TOX3, SMARCE1, HMG20B, HMGB3, HMGA2, HMGA1, ARX, HOXA11,MEOX1, DLX6, ISL1, HOXC8, BARX2, ALX4, GSC2, DLX3, PITX1, HOXA9, HOXA10,LHX5, LASS4, ZFHX4, SIX4, VSX1, ADNP, RHOXF1, MEIS3, PBX4, DLX5, HOXA1,HOXA2, HOXA3, HOXA5, HOXA6, HOXA13, EVX1, NOBOX, MEOX2, LHX2, LHX6,LHX3, TLX1, PITX3, HOXB6, HNF1B, DLX4, SEBOX, VTN, PHOX2B, NKX3-2, DBX1,NANOG, IRX4, CDX1, TLX2, DLX2, VAX2, PRRX1, TGIF2, VSX2, NKX2-3, HOXB8,HOXB5, HOXB7, HOXB3, HOXB1, MSX2, LHX4, HOXA7, HOXC13, HOXC11, HOXC12,ESX1, BARHL1, NKX2-4, NKX2-2, SIX1, HOXD1, HOXD3, HOXD9, HOXD10, HOXD11,HOXD13, MNX1, CDX4, BARX1, RHOXF2, LHX1, GSC, MEIS2, RAX, EMX1, NKX2-8,NKX2-1, HLX, LMX1B, SIX3, LBX1, PDX1, LASS5, ZFHX3, BARHL2, LHX9, LASS2,MEIS1, DLX1, HMBOX1, ZEB1, VAX1, NKX6-2, VENTX, HHEX, TGIF2 LX, LASS3,ALX3, HOXB13, IRX6, ISL2, PKNOX1, LHX8, LMX1A, EN1, MSX1, NKX6-1, HESX1,PITX2, TLX3, EN2, UNCX, GBX1, NKX6-3, ZHX1, HDX, PHOX2A, PKNOX2, CDX2,DRGX, NKX3-1, PBX3, PRRX2, GBX2, SHOX2, GSX1, HOXD4, HOXD12, EMX2, IRX1,IRX2, SIX2, HOXB9, HOPX, OTP, LASS6, HOXC5, HOXB2, RAX2, EVX2, ZHX3,PROP1, ISX, HOXD8, TGIF2 LY, IRX5, SIX5, TGIF1, IRX3, ZHX2, LBX2,NKX2-6, ALX1, GSX2, HOXC9, HOXC10, HOXB4, NKX2-5, SIX6, MIXL1, DBX2,PBX1, SHOX, ARGFX, HMX3, HMX2, BSX, HOXA4, DMBX1, HOXC6, HOXC4, RHOXF2B,PBX2, DUXA, DPRX, LEUTX, NOTO, HOMEZ, HMX1, DUX4 L5, DUX4 L2, DUX4 L3,DUX4 L6, NKX1-1, HNF1A, HSF4, HSFY2, HSFX1, HSFX2, HSFY1, HSF1, LCORL,LCOR, IRF6, IRF1, IRF3, IRF5, IRF4, IRF8, IRF2, IRF7, IRF9, MBD3, BAZ2B,MBD4, SETDB2, MBD1, MECP2, SETDB1, MBD2, BAZ2A, SMAD7, SMAD5, SMAD9,SMAD6, SMAD4, SMAD3, SMAD1, SMAD2, ZZZ3, RCOR1, CDC5L, MYBL2, DNAJC2,TADA2A, RCOR3, MYB, TERF2, DMTF1, DNAJC1, NCOR1, TERF1, MIER3, MYSM1,SNAPC4, RCOR2, TADA2B, MYBL1, TERF1P2, NCOR2, CCDC79, SMARCC1, SMARCC2,TTF1, C11orf9, NFYA, NFYC, NFYB, NRF1, NR4A3, NR4A1, NR4A2, ESR1, NR0B2,NR0B1, PREB, EAF2, SPZ1, TP63, TP73, TP53, PAX6, PAX7, PAX2, PAX4, PAX8,PAX1, PAX3, PAX5, PAX9, SUB1, POU2F2, POU1F1, POU4F3, POU6F2, POU2F3,POU2F1, POU4F2, POU4F1, POU6F1, POU3F2, POU3F1, POU3F4, POU3F3, POU5F1,POU5F1B, PPARD, PPARG, PPARA, PGR, PROX1, PROX2, NR2E1, NR5A2, NR2C1,NR5A1, NR6A1, ESRRA, NR2C2, RFX3, RFX2, RFX4, RFX1, RFX5, RFX7, RFX6,RFX8, NFATC3, NFKB2, NFATC4, NFATC2, NFAT5, RELB, NFKB1, NFATC1, REL,RELA, RORA, RORC, NR1D2, RORB, RUNX3, RUNX1, SP100, SP140, GMEB2, SP110,AIRE, GMEB1, DEAF1, SP140 L, LOC729991-MEF2B, MEF2A, SRF, MEF2D, MEF2B,STAT1, STAT5A, STAT4, STAT6, STAT3, STAT2, STAT5B, TBX21, TBX5, TBX15,TBX18, TBX2, TBX4, TBX22, TBX3, TBR1, TBX19, TBX6, EOMES, T, TBX20,TBX10, MGA, TBX1, TEAD3, TEAD2, TEAD1, TEAD4, CREBL2, NFE2 L3, CREB3 L3,FOSL2, NFE2 L1, CREM, DBP, CREB3, HLF, BACH2, ATF2, NFE2 L2, ATF6,CREB1, ATF1, NFE2, FOSB, ATF4, NRL, JUND, JDP2, CREB3 L4, BATF, BACH1,CREB3 L1, NFIL3, TEF, BATF2, ATFS, FOS, JUNB, DDIT3, FOSL1, JUN, MAF,CREB3 L2, MAFA, MAFF, MAFG, MAFK, MAFB, ATF6B, CRX, OTX1, OTX2, THAP3,THAP10, THAP1, PRKRIR, THAP8, THAP9, THAP11, THAP2, THAP6, THAP4, THAP5,THAP7, NR1H4, NR2E3, RARB, HNF4A, VDR, ESRRB, THRA, NR1D1, RARA, ESR2,NR1I3, NR1I2, THRB, NR3C2, HNF4G, RARG, RXRA, ESRRG, RXRB, TSC22D1,TSC22D3, TSC22D4, TSC22D2, TULP3, TULP2, TULP1, TULP4, TUB, ZBTB33,ZBTB32, ZBTB11, MYNN, ZBTB25, PATZ1, ZBTB16, ZBTB24, BCL6, ZBTB47,ZBTB17, ZBTB45, GZF1, ZBTB1, ZBTB46, ZBTB8A, ZBTB7B, BCL6B, ZBTB49,ZBTB43, HIC2, ZBTB26, ZNF131, ZNF295, ZBTB4, ZBTB34, ZBTB38, HIC1,ZBTB41, ZBTB7A, ZNF238, ZBTB42, ZBTB2, ZBTB20, ZBTB40, ZBTB7C, ZBTB37,ZBTB3, ZBTB6, ZBTB44, ZFP161, ZBTB12, ZBTB48, ZBTB10, ZBED4, ZBED3,ZBED2, C11orf95, ZBED1, IKZF5, ZNF821, ZNF451, ZNF195, ZFX, ZNF263,ZNF200, HIVEP2, WIZ, ZNF582, SNAI2, ZFP64, IKZF2, ZIC2, ZNF800, PRDM1,PRDM6, ZFP112, ZNF275, ZNF76, ZFAT, KLF6, ZFY, ZXDC, GLI2, ZNF532,ZNF37A, ZNF510, ZNF506, ZNF324, ZNF671, ZNF416, ZNF586, ZNF446, ZNF8,ZNF264, REST, MECOM, ZNF213, ZNF343, ZNF302, ZNF268, ZNF10, HIVEP1,ZNF184, MZF1, SALL4, ZNF516, KLF8, KLF5, ZNF629, ZNF423, CTCF, ZNF500,ZNF174, SALL1, MAZ, ZNF419, OVOL3, ZNF175, ZNF14, ZNF574, ZNF85, SP4,ZKSCAN1, GLI3, GLIS3, KLF3, PRDM4, GLI1, PRDM13, ZNF142, PRDM2, ZNF684,ZNF541, KLF7, PLAGL1, ZNF430, KLF12, KLF9, ZNF410, BCL11A, EGR1, ZFP30,TSHZ3, ZNF549, ZSCAN18, ZNF211, ZNF639, ZSCAN20, GTF3A, ZNF205, ZNF644,EGR2, IKZF4, CTCFL, ZNF831, SNAI1, ZNF576, ZNF45, TRERF1, ZNF391, RREB1,ZNF133, OVOL2, ZNF436, PLAGL2, GLIS2, ZNF384, ZNF484, HIVEP3, BCL11B,KLF2, ZNF780B, FEZF1, KLF16, ZSCAN10, ZNF557, ZNF337, PRDM12, ZNF317,ZNF426, ZNF331, ZNF236, ZNF341, ZNF227, ZNF141, ZNF304, ZSCANSA, ZNF132,ZNF20, EGR4, ZNF670, VEZF1, KLF4, ZFP37, ZNF189, ZNF193, ZNF280D, PRDM5,ZNF740, ZIC5, ZSCAN29, ZNF710, ZNF434, ZNF287, ZIM3, PRDM15, ZFP14,ZNF787, ZNF473, ZNF614, PRDM16, ZNF697, ZNF687, OSR1, ZNF514, ZNF660,ZNF300, RBAK, ZNF92, ZNF157, ZNF182, ZNF41, ZNF711, PRDM14, ZNF7,ZNF214, ZNF215, SALL3, ZNF827, ZNF547, ZNF773, ZNF776, ZNF256, ZSCAN1,ZNF837, PRDM8, ZNF117, ZIC1, FEZF2, ZNF599, ZNF18, KLF10, ZKSCAN2,ZNF689, ZIC3, ZNF19, ZSCAN12, ZNF276, ZNF283, ZNF221, ZNF225, ZNF230,ZNF222, ZNF234, ZNF233, ZNF235, ZNF362, ZNF208, ZNF714, ZNF394, ZNF333,ZNF382, IKZF3, ZNF577, ZNF653, ZNF75A, GFI1, ZNF281, ZNF496, ZNF2,ZNF513, ZNF148, KLF15, ZNF691, ZNF589, PRDM9, ZNF12, SP8, OSR2, ZNF367,ZNF22, GFI1B, ZNF219, SALL2, ZNF319, ZNF202, ZNF143, ZNF3, ZSCAN21,ZNF606, SP2, ZNF91, ZNF23, ZNF226, ZNF229, ZNF180, ZNF668, ZNF646,ZNF641, ZNF610, ZNF528, ZNF701, ZNF526, ZNF146, ZNF444, ZNF83, ZNF558,ZNF232, E4F1, ZNF597, INSM2, ZNF30, ZNF507, ZNF354A, ZEB2, ZNF32, KLF13,ZFPM2, ZNF764, ZNF768, ZNF35, ZNF778, ZNF212, ZNF282, PRDM10, SP7,SCRT1, ZNF16, ZNF296, ZNF160, ZNF415, ZNF672, ZNF692, ZNF439, ZNF440,ZNF581, ZNF524, ZNF562, ZNF561, ZNF584, ZNF274, ZIK1, ZNF540, ZNF570,KLF17, ZNF217, ZNF57, ZNF556, ZNF554, KLF11, HINFP, ZNF24, ZNF596,OVOL1, SP3, ZNF621, ZNF680, BNC2, ZNF483, ZNF449, INSM1, ZNF417, ZNF791,ZNF80, GLIS1, ZNF497, KLF14, ZNF266, ZIC4, ZNF408, ZNF519, ZNF25, ZNF77,ZNF169, ZNF613, ZNF683, ZNF135, ZSCAN2, ZNF575, ZNF491, ZNF620, ZNF619,ZNF354C, ZNF114, ZNF366, ZNF454, ZNF543, ZNF354B, ZNF223, ZNF713,ZNF852, ZNF552, ZFP42, ZNF664, EGR3, ZFPM1, ZNF784, ZNF648, FIZ1,ZNF771, TSHZ1, ZNF48, ZNF816, ZNF571, ZSCAN4, ZNF594, ZFP3, ZNF443,ZNF792, ZNF572, ZNF707, ZNF746, ZNF322A, ZNF467, ZNF678, ZFP41, HKR1,PLAG1, ZNF329, ZNF101, ZNF716, ZNF708, ZSCAN22, ZNF662, ZNF320, ZNF623,ZNF530, ZNF285, ZFP1, WT1, ZFP90, ZNF479, ZNF445, ZNF74, SP1, SNAI3,ZNF696, IKZF1, ZNF267, ZNF566, ZNF224, ZNF529, ZNF284, ZNF749, ZNF17,ZNF555, ZNF75D, ZNF501, ZNF197, ZNF396, ZFP91, ZNF732, ZNF397, ZSCAN30,ZNF546, ZNF286A, ZKSCAN4, ZNF70, ZNF643, ZNF642, ZSCAN23, ZNF490,ZNF626, ZNF793, ZNF383, ZNF669, ZNF559, ZNF177, ZNF548, MTF1, ZNF322B,ZNF563, ZNF292, ZNF567, SP6, ZNF573, ZNF527, ZNF33A, ZNF600, ZKSCAN3,ZNF676, ZNF699, ZNF250, ZNF79, ZNF681, ZNF766, ZNF107, ZNF471, ZNF836,ZNF493, ZNF167, ZNF565, ZNF34, ZNF781, ZNF140, ZNF774, ZNF658, ZNF765,ZNF124, ZNF569, ZNF777, ZNF775, ZNF799, ZNF782, ZNF846, ZNF136, ZKSCANS,ZNF502, ZFP62, ZNF33B, ZNF512B, ZNF431, ZNF418, ZNF700, ZNF239, ZSCAN16,ZFP28, ZNF705A, ZNF585A, ZNF138, ZNF429, ZNF470, ZNF100, ZNF398, ZNF498,ZNF441, ZNF420, ZNF763, ZNF679, ZNF682, ZNF772, ZNF257, ZNF785, ZSCAN5B,ZNF165, ZNF655, ZNF98, ZNF786, ZNF517, ZNF675, ZNF860, ZNF628, ZNF665,ZNF624, ZNF841, ZNF615, ZNF350, ZNF432, ZNF433, ZNF460, ZNF81, ZNF780A,ZNF461, ZNF181, LOC100287841, ZNF44, ZNF790, ZNF677, ZNF823, ZNF311,ZNF347, ZNF71, ZNF121, ZNF335, ZNF560, ZNF273, ZNF84, ZNF667, ZNF649,ZNF248, ZNF544, ZNF770, ZNF737, ZNF251, ZNF607, ZNF334, ZXDA, ZNF485,ZIM2, PEG3, ZNF192, ZNF442, ZNF813, ZNF26, ZNF69, ZNF583, ZNF568, ZXDB,ZNF480, ZNF587, ZNF808, ZNF43, ZNF28, ZNF627, ZNF789, ZNF536, ZNF534,ZNF652, ZNF521, ZNF358, ZFP2, SP5, ZNF814, ZNF551, ZNF805, ZSCAN5C,ZNF468, ZNF616, ZFP57, ZNF155, ZNF783, ZNF425, ZNF580, ZNF611, ZNF254,ZNF625, ZNF134, ZNF845, ZNF99, ZNF253, ZNF90, ZNF93, ZNF486, REPIN1,LOC100131539, ZNF705D, LOC100132396, ZNF705G, SCRT2, ZNF407, SP9,ZNF579, ZNF880, ZNF630, ZNF844, ZNF469, ZNF717, ZNF865, ZNF492, ZNF688,YY2, ZNF878, ZNF879, ZNF736, ZNF323, ZNF709, ZNF512, ZNF585B, ZNF154,ZNF324B, ZNF564, ZFP82, GLI4, ZNF674, ZNF345, ZNF550, KLF1, YY1, MYST2,ST18, L3MBTL4, MYT1 L, MYT1, L3MBTL1, MTA3, GATA1, TRPS1, GATA3, GATAS,GATA4, GATA6, GATAD2B, GATAD1, GATA2, MTA1, ZGLP1, MTA2, RERE, C16orf5,LITAF, PIAS1, PIAS2, PIAS4, ZMIZ1, ZMIZ2, PIAS3, RNF138, NFX1, NFXL1, orany combinations thereof.

In some embodiments, cells are manipulated (e.g., converted ordifferentiated) from one cell type to another. In some embodiments, apancreatic cell is manipulated into a beta islet cell. In someembodiments, a fibroblast is manipulated into an iPS cell. In someembodiments, a preadipocyte is manipulated into a brown fat cell. Otherexemplary cells include, e.g., muscle cells, neural cells, leukocytes,and lymphocytes.

In some embodiments, the cell is a diseased or mutant-bearing cell. Suchcells can be manipulated to treat the disease, e.g., to correct amutation, or to alter the phenotyope of the cell, e.g., to inhibit thegrowth of a cancer cell. For example, a cell is associated with one ormore diseases or conditions described herein.

In some embodiments, the manipulated cell is a normal cell.

In some embodiments, the manipulated cell is a stem cell or progenitorcell (e.g., iPS, embryonic, hematopoietic, adipose, germline, lung, orneural stem or progenitor cells). In some embodiments, the manipulatedcell can be a cell from any of the three germ layers (i.e. mesodermal,endodermal or ectodermal. In some embodiments, the manipulated cell canbe from an extraembryonic tissue, for example, from the placenta.

In some embodiments, the cell being manipulated is selected fromfibroblasts, monocytic-precursors, B cells, exocrine cells, pancreaticprogenitors, endocrine progenitors, hepatoblasts, myoblasts, orpreadipocytes. In some embodiments, the cell is manipulated (e.g.,converted or differentiated) into muscle cells, erythroid-megakaryocyticcells, eosinophils, iPS cells, macrophages, T cells, islet beta-cells,neurons, cardiomyocytes, blood cells, endocrine progenitors, exocrineprogenitors, ductal cells, acinar cells, alpha cells, beta cells, deltacells, PP cells, hepatocytes, cholangiocytes, angioblast, mesoangioblastor brown adipocytes.

In some embodiments, the cell is a muscle cell, erythroid-megakaryocyticcell,

eosinophil, iPS cell, macrophage, T cell, islet beta-cell, neuron,cardiomyocyte, blood cell, endocrine progenitor, exocrine progenitor,ductal cell, acinar cell, alpha cell, beta cell, delta cell, PP cell,hepatocyte, cholangiocyte, or white or brown adipocyte.

In some embodiments, the cell is a precursor cell, a pluripotent cell, atotipotent cell, an adult stem cell, an inner cell mass cell, anembryonic stem cell, or an iPS cell.

In some embodiments, the manipulated cell is a cancer cell. In someembodiments, the cancer cell can be a lung cancer cell, a breast cancercell, a skin cancer cell, a brain cancer cell, a pancreatic cancer cell,a hematopoietic cancer cell, a liver cancer cell, a kidney cancer cell,an ovarian cancer cell, a prostate cancer cell, a skin cancer cell.

In some embodiments, the cell is a muscle cell, erythroid-megakaryocyticcell, eosinophil, iPS cell, macrophage, T cell, islet beta-cell, neuron,cardiomyocyte, blood cell, endocrine progenitor, exocrine progenitor,ductal cell, acinar cell, alpha cell, beta cell, delta cell, PP cell,hepatocyte, cholangiocyte, or white or brown adipocyte.

Administration of DNA-PK Inhibitors and Gene-Editing System to a Cell(s)

Administering to a cell(s) a genome editing system and a DNA-PKinhibitor can be performed by any method known in the art. Theadministering can be in vitro, ex vivo or in vivo. The administering toa cell(s) a genome editing system and a DNA-PK inhibitor can occursimultaneously or sequentially. In some embodiments, the administeringresults in the DNA-PK inhibitor and the genome editing system componentsto enter the cell membrane. In some embodiments, the administeringresults in the DNA-PK inhibitor and the genome editing system componentsto enter into the cell nucleus. In some embodiments, the administeringincludes incubating the cell in the presence of the DNA-PK inhibitor andgenome editing system.

The gene editing system can be administered to a cell(s) by any methodknown in the art. For example, any nucleic acid or protein deliverymethods known in the art can be used. The gene editing system isadministered (e.g., delivered) to a cell by way of a nucleic acidencoding the gene editing system components. The gene editing system canbe administered to a cell by either viral vectors or non-viral vectors.In some embodiments, viral vectors are used. The viral vectors can beretroviral (e.g. murine leukemia, HIV, or lentiviral) or DNA viruses(e.g. adenovirus, herpes simplex, and adeno-associated). In someembodiments, transfection methods (e.g. non-viral delivery methods) areused to introduce the genome editing system into a cell. Transfectionmethods include contacting the cell with DEAE-Dextran, calciumphosphate, liposomes or electroporation of a plasmid into a cell.Additional methods of non-viral delivery include electroporation,lipofection, microinjection, biolistics, virosomes, liposomes,immunoliposomes, polycation or lipid: nucleic acid conjugates, nakedDNA, naked RNA, artificial virions, and agent-enhanced uptake of DNA.Sonoporation using, e.g., the Sonitron 2000 system (Rich-Mar) can alsobe used for delivery of nucleic acids. In some embodiments, one or morenucleic acids are delivered as mRNA. In some embodiments, capped mRNAsare used to increase translational efficiency and/or mRNA stability. Insome embodiments, ARCA (anti-reverse cap analog) caps or variantsthereof are used. See US patents US7074596 and U.S. Pat. No. 8,153,773.

In embodiments, the endonuclease (e.g. Cas, Cpf1 and the like) and thegRNA, are transcribed from DNA.

In embodiments, the endonuclease (e.g. Cas, Cpf1 and the like) istranscribed from DNA and the gRNA is provided as RNA.

In embodiments, the endonuclease (e.g. Cas, Cpf1 and the like) and thegRNA are provided as RNA.

In embodiments, the endonuclease (e.g. Cas, Cpf1 and the like) isprovided as a protein and the gRNA is provided as DNA.

In embodiments, the endonuclease (e.g. Cas, Cpf1 and the like) isprovided as protein and the gRNA is provided as RNA.

Additional nucleic acid delivery systems include those provided by AmaxaBiosystems (Cologne, Germany), Maxcyte, Inc. (Rockville, Md.), BTXMolecular Delivery Systems (Holliston, Mass.) and CopernicusTherapeutics Inc, (see for example U.S. Pat. No. 6,008,336). Lipofectionis described in e.g., U.S. Pat. Nos. 5,049,386; 4,946,787; and4,897,355) and lipofection reagents are sold commercially (e.g.,Transfectam™ and Lipofectin™ and Lipofectamine™ RNAiMAX). Cationic andneutral lipids that are suitable for efficient receptor-recognitionlipofection of polynucleotides include those of Feigner, WO 91/17424, WO91/16024. Delivery can be to cells (ex vivo administration) or targettissues (in vivo administration).

The preparation of lipid:nucleic acid complexes, including targetedliposomes such as immunolipid complexes, is well known to one of skillin the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese etal., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem.5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gaoet al., Gene Therapy 2:710-722 (1995);

Additional methods of delivery include the use of packaging the nucleicacids to be delivered into EnGenelC delivery vehicles (EDVs). These EDVsare specifically delivered to target tissues using bispecific antibodieswhere one arm of the antibody has specificity for the target tissue andthe other has specificity for the EDV. The antibody brings the EDVs tothe target cell surface and then the EDV is brought into the cell byendocytosis. Once in the cell, the contents are released (see MacDiarmidet al (2009) Nature Biotechnology 27(7):643) Ahmad et al., Cancer Res.52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871,4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).

In some embodiments, the transfection can be transient in which thetransfected genome editing system containing plasmid enters the nucleusbut does not become incorporated into the genome of the cell duringreplication. The transfection can be stable in which the transfectedplasmid will become integrated into a genomic region of the cell.

In some embodiments in which transient expression is used, adenoviralbased systems can be used. Adenoviral based vectors are capable of veryhigh transduction efficiency in many cell types and do not require celldivision. With such vectors, high titer and high levels of expressionhave been obtained. This vector can be produced in large quantities in arelatively simple system. Adeno-associated virus (“AAV”) vectors arealso used to transduce cells with target nucleic acids, e.g., in the invitro production of nucleic acids and peptides, and for in vivo and exvivo gene therapy procedures (see, e.g., West et al., Virology 160:38-47(1987); U.S. Pat. No. 4,797,368; WO 93/24641; Kotin, Human Gene Therapy5:793-801 (1994); Muzyczka, J. Clin. Invest. 94: 1351 (1994).Construction of recombinant AAV vectors are described in a number ofpublications, including U.S. Pat. No. 5,173,414; Tratschin et al., Mol.Cell. Biol. 5:3251-3260 (1985); Tratschin, et al., Mol Cell. Biol.4:2072-2081 (1984); Hermonat & Muzyczka, PNAS 81:6466-6470 (1984); andSamulski et al., J. Virol 63:03822-3828 (1989).

In some embodiments, the administering to a cell(s) of a DNA-PKinhibitor is performed by culturing an isolated cell(s) in the presenceof the DNA-PK inhibitor and any suitable medium that allows for theDNA-PK inhibitor to enter the cell membrane and/or the cell nucleus.

In some embodiments, the DNA-PK inhibitors are administered to a cell(s) in vitro, in vivo or ex vivo. In some embodiment, the DNA-PKinhibitor is contacted with a cell(s) for about 5 hours, 10 hours, 15hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 30hours, 35 hours, 40 hours, 45 hours, 50 hours, 55 hours, 60 hours, 65hours, 70 hours, 85 hours, 90 hours, 100 hours, 125 hours, 150 hours,200 hours, or for any period of time in between. In some embodiments,the DNA-PK inhibitor is contacted with a cell(s) for about 1.5 weeks,2.0 weeks, 2.5 weeks, 3.0 weeks, 3.5 weeks, 4 weeks, or any period oftime in between. The DNA-PK inhibitor may be re-administered with cellculture medium changes. The DNA-PK inhibitor can be contacted with thecell either before, during or after introduction of genome editingsystem components.

In some embodiments, the DNA-PK inhibitor is administered to a cell(s)at a concentration of about 0.1 μM, 0.25 μM, 0.5 μM, 0.75 μM, 1.0 μM,1.25 μM, 1.50 μM, 1.75 μM, 2.0 μM, 2.5 μM, 3.0 μM, 3.5 μM, 4.0 μM, 4.5μM, 5.0 μM, 5.5 μM, 6.0 μM, 6.5 μM, 7.0 μM, 7.5 μM, 8.0 μM, 8.5 μM, 9.0μM, 9.5 μM, 10 μM, 10.5 μM, 11.0 μM, 11.5 μM, 12μM, or anyconcentrations in between. The DNA-PK inhibitor concentration can bemodified during the course of administration.

In some embodiments, the gene-editing components are delivered into acell(s) by one or more vectors or in the form of RNA, mRNA or in thecase of the endonuclease component as purified protein or mRNA (e.g.Cas9 protein). The one or more vectors can include viral vectors,plasmids or ssDNAs. Viral vectors can include retroviral, lentiviral,adenoviral, adeno-associated, and herpes simplex viral vectors, or anycombinations thereof. In some embodiments, the gene-editing componentsare delivered via RNA or synthetic RNA.

In some embodiments, administration of the DNA-PK inhibitors to a cellalong with a gene-editing system results in increased amounts ofhomologous directed repair gene-editing outcome in comparison to abaseline condition in which the cell is not administered a DNA-PKinhibitor. In some embodiments, administration of the DNA-PK inhibitorsto a cell(s) along with a gene-editing system results in suppression ofindels (from NHEJ) either on-target or off-target. In some embodiments,administration of the DNA-PK inhibitors to a cell(s) along with agene-editing system results in increased or decreased expression of agene of interest. Administration of the DNA-PK inhibitors to a cell(s)along with a gene-editing system can result in the expression of a genenot endogenous to a cell. In some embodiments, administration of theDNA-PK inhibitors to a cell(s) along with a gene-editing system resultsin the complete or partial removal, or a modification of a gene from acell(s). In some embodiments, administration of the DNA-PK inhibitors toa cell(s) along with gene-editing system result(s) in the complete orpartial removal, or a modification of an intron and/or an exon in acell(s). In some embodiments, administration of the DNA-PK inhibitors toa cell(s) along with gene-editing system result(s) in the complete orpartial removal, or a modification of a non-coding region in a cell(s).In some embodiments, administration of the DNA-PK inhibitors to a cellalong with gene-editing system result(s) in simultaneous or sequential,complete or partial removal, or a modification of a coding and/ornon-coding genetic region in a cell(s). In some embodiments,administration of the DNA-PK inhibitors to a cell(s) along withgene-editing system results in simultaneous or sequential, complete orpartial removal, or a modification of a coding and/or non-coding geneticregion in a cell(s), including extrachromosomal DNA or RNA. TheExtrachromosomal DNA can be mitochondrial DNA, chloroplast DNA,extrachromosomal circular DNA, or viral extra chromosomal DNA.

In some embodiments, administration of DNA-PK inhibitors to a cell alongwith genome editing system results in increased expression or decreasedexpression of a gene of interest. In some embodiments, the increase ordecrease in expression of a gene of interest can be about or between,2.5%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% in comparison to abaseline condition in which the cell is not administered a DNA-PKinhibitor. In some embodiments, the increase or decrease of a gene ofinterest can be about or between, 0.5-fold, 1.0-fold, 1.5-fold,2.0-fold, 2.5-fold, 3.0-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold or10-fold in comparison to the baseline expression level in which the cellis not administered a DNA-PK inhibitor.

In some embodiments, administration of DNA-PK inhibitors to a cell alongwith a genome editing system results in an increase in genome editing.In some embodiments, the increase in genome editing can be about orbetween 2.5%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% incomparison to a baseline condition in which the cell is not administereda DNA-PK inhibitor. In some embodiments, the increase in genome editingcan be about or between 0.5-fold, 1.0-fold, 1.5-fold, 2.0-fold,2.5-fold, 3.0-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold or 10-fold incomparison to the baseline expression level in which the cell is notadministered a DNA-PK inhibitor.

In some embodiments, administration of a DNA-PK inhibitor and a geneediting system to a cell population results in greater cell survival incomparison to a baseline condition in which a cell population onlyadministered a gene editing system and is not administered a DNA-PKinhibitor. In some embodiments, the DNA-PK inhibitor that results ingreater cell survival is a compound of formula (III-E-1) or (III-E-2),or pharmaceutically acceptable salts thereof.

In some embodiments, the cell is synchronized at the S or G2 cell cyclephase, either before, after or during administration of the DNA-PKinhibitor. In some embodiments, the cell is synchronized at the S or G2cell cycle phase, either before, after or during introduction of thegene-editing components. Synchronization of the cell at the S or G2 cellcycle phase can be achieved by any method known in the art. As anon-limiting example, agents that can be used to synchronize a cell atthe S or G2 cell cycle phase include aphidicolin, dyroxyurea,lovastatin, mimosine, nocodazole, thymidine, or any combinationsthereof. (See, Lin et al. Elife. 2014 Dec. 15; 32014). In someembodiments, the agents for cell synchronization can be administered atany time during the gene-editing process.

In some embodiments, the DNA-PK inhibitor and/or the genome editingsystem can be included in a container, pack, or dispenser together withinstructions for use. In some embodiments, the DNA-PK inhibitor agentand/or the genome editing system included in a container, pack ordispenser together with instructions for use is a kit.

In some embodiments, the DNA-PK inhibitors and/or the genome editingsystem are included in a kit with instructions for use. The kit cancontain any genome editing system, and/or DNA-PK inhibitor andinstructions for use. In some embodiments the DNA-PK inhibitor is any ofcompounds represented by Structural Formula (III-E-1) or (III-E-2). Insome embodiments, the genome editing system is a selected from ameganuclease based system, a zinc finger nuclease (ZFN) based system, aTranscription Activator-Like Effector-based Nuclease (TALEN) system, aCRISPR-based system, or a NgAgo-based system. The genome editing systemcan be provided in the kit in any form, for example as a plasmid,vector, DNA, or RNA construct.

In some embodiments, the DNA-PK inhibitor and/or a genome editing systemis administered in vivo. The DNA-PK inhibitor and the gene-editingsystem is formulated to be compatible with its intended route ofadministration. Examples of routes of administration are describedabove.

In some embodiments, the formulation can also contain more than oneactive compound as necessary for the particular indication beingtreated, for example, those with complementary activities that do notadversely affect each other. Alternatively, or in addition, thecomposition can comprise an agent that enhances its function, such as,for example, a cytotoxic agent, cytokine, chemotherapeutic agent, orgrowth-inhibitory agent. Such molecules are suitably present incombination in amounts that are effective for the purpose intended.

In some embodiments, the DNA-PK inhibitor agent and/or the genomeediting system are administered in combination therapy, i.e., combinedwith other agents, e.g., therapeutic agents, that are useful fortreating pathological conditions or disorders, such as various forms ofcancer and inflammatory diseases. The term “in combination” in thiscontext means that the agents are given substantially contemporaneously,either simultaneously or sequentially. If given sequentially, at theonset of administration of the second compound, the first of the twocompounds is preferably still detectable at effective concentrations atthe site of treatment.

Genome Editing Screening Methods

Any method known in the art can be used to screen cells forgenome-editing efficiency, including the efficiency of NHEJ and/or HDR.For example, screening methods can include PCR based amplification oftargeted regions followed by sequencing or deep sequencing of theamplified regions to confirm genome editing. PCR genotyping permits thequantification and ranking of compounds in stimulating HDR. Otherscreening methods can include next-generation sequencing. See, forexample Bell et al., “A high-throughput screening strategy for detectingCRISPR-Cas9 induced mutations using next-generation sequencing,” BMCGenomics, 15:1002 (2014).

PCR primers can be engineered to selectively amplify both unmodified andmodified genetic regions, resulting in amplicons of different lengthsdepending on the genetic modification status. The amplicons can then beresolved on a gel, and the HDR efficiency estimated by densitometryusing a Bio-Imager. Alternatively, a new PCR technology, the rapiddigital droplet PCR (DDPCR) can be used to simultaneously measure HDRand NHEJ events in genome-edited samples. See, for example, Miyaoka etal., “Systematic quantification of HDR and NHEJ reveals effectrs oflocus, nuclease, and cell type on genome-editin,” Scientific Reports, 6,2016. Other methods that can be used for screening cells for genomicmodiciations including, Sanger sequencing, deep sequencing, and RT-PCR.15

Preparation of Compounds of the Invention

As used herein, all abbreviations, symbols and conventions areconsistent with those used in the contemporary scientific literature.See, e.g., Janet S. Dodd, ed., The ACS Style Guide: A Manual for Authorsand Editors, 2nd Ed., Washington, D.C.: American Chemical Society, 1997.The following definitions describe terms and abbreviations used herein:

-   BPin pinacol boronate ester-   Brine a saturated NaCl solution in water-   DCM dichloromethane-   DIAD diisopropylazodicarboxylate-   DIEA diisopropylethylamine-   DMA dimethylacetamide-   DMF dimethylformamide-   DMSO dimethylsulfoxide-   DTT dithiothreitol-   ESMS electrospray mass spectrometry-   Et₂O ethyl ether-   EtOAc ethyl acetate-   EtOH ethyl alcohol-   HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-   HPLC high performance liquid chromatography-   IPA isopropanol-   LAH lithium aluminum hydride-   LC-MS liquid chromatography-mass spectrometry-   LDA lithium diisoproylethylamide-   Me methyl-   MeOH methanol-   MsCl methanesulfonyl chloride-   MTBE methyl t-butyl ether-   NMP N-methylpyrrolidine-   Pd₂(dba)₃ tris(dibenzylideneacetone)dipalladium(0)-   Pd(dppf)Cl₂ 1,1′bis(diphenylphosphino)-ferrocene dichloro-palladium-   PG protecting group-   Ph phenyl-   (rac)-BINAP racemic 2,2′-bis(diphenylphosphino)-1,1′-binaphthyl-   RockPhos    di-tert-butyl(2′,4′,6′-triisopropyl-3,6-dimethoxy-[1,1′-biphenyl]-2-yl)phosphine-   RT or rt room temperature-   SFC supercritical fluid chromatography-   SPhos 2-dicyclohexylphosphino-2′,6′-dimethoxybiphenyl-   TBAI tetrabutylammonium iodide-   tBu tertiary butyl-   THF tetrahydrofuran-   TEA triethylamine-   TMEDA tetramethylethylenediamine-   VPhos    [3-(2-dicyclohexylphosphanylphenyl)-2,4-dimethoxy-phenyl]sulfonyloxysodium

General Synthetic Procedures

In general, the compounds of this invention may be prepared by methodsdescribed herein or by other methods known to those skilled in the art.

EXAMPLE 1 General Preparation of the Compounds of Formula G

Compounds of formula (III-E-1) or (III-E-2), wherein X is NH can beprepared as outlined below in Scheme 1. Accordingly, as shown in step1-i of Scheme 1, heteroaryl compounds of formula A can be reacted withmorpholine or a morpholine analog by heating the mixture in a polar,non-protic solvent to produce compounds of formula B. Utilizing apalladium-catalyzed, phosphine ligand-assisted Buchwald/Hartwig-typecoupling, as shown in step 1-ii of Scheme 1, a compound of formula B canbe reacted with aminocyclohexanes of formula C to produce compounds offormula D, wherein R¹ and R² are as described elsewhere herein. In oneexample, when monoprotected meso cyclohexane-1,4-diamines of formula Eare prepared, removal of the protecting group forms compounds of formulaF, as shown in step 1-iii of Scheme 1. The resulting free amine can thenbe reacted with various moieties having groups reactive towards amines(e.g., R^(1a)-L, where L is a leaving group such as chloro, bromo, iodo,toluenesulfonate, methanesulfonate, or trifluoromethanesulfonate; orwhere L is a reactive carbonyl-containing moiety such as an active esteror an isocyanato group) to produce a compound of formula G, as shown instep 1-iv of Scheme 1.

EXAMPLE 2 General Preparation of the Compounds of Formula M. N. R. and S

Compounds of (III-E-1) or (III-E-2), wherein X is O can be prepared asoutlined below in Schemes 2a and 2b. Accordingly, as shown in step 2-iof Scheme 2a, the hydroxyl group of heteroaryl compounds of formula Hcan be protected to produce compounds of formula J, which can then bereacted with morpholine or a morpholine analog by heating the mixture ina polar, non-protic solvent to produce compounds of formula K afterremoval of the protecting group, as shown in steps 2-ii and 2-iii ofScheme 2a. Subsequently, as shown in step 2-iv of Scheme 2a, a compoundof formula K can be reacted with a compound of formula L underconditions sufficient to affect the SN2 displacement of its leavinggroup (e.g., where L is a leaving group such as chloro, bromo, iodo,toluenesulfonate, methanesulfonate, or trifluoromethanesulfonate) toproduce a compound of formula M or formula M, depending on whether R¹ orR² is hydrogen. In those instances when R¹ or R² are protected nitrogenor oxygen moieties, compounds of the invention can be produced byremoval of the protecting group and subsequent synthetic manipulation ofthe resulting free amine/alcohol.

Alternatively .formula P, which can subsequently be reacted withmorpholine or a morpholine analog to produce a compound of formula M orformula N.

Alternatively, as shown in Scheme 2c, compounds of the invention inwhich Ring B is a dihydropyran ring can be prepared by reactingcompounds of formula Q with dialkyl (3,6-dihydro-2H-pyran-4-yl)boronatesto produce compounds of formula R. Compounds of formula R can then besubsequently reduced to form compounds of formula S.

EXAMPLE 3 Preparation of ethyl(4-((7-morpholinoquinoxalin-5-yl)amino)cyclohexyl)carbamate (Compound 6)andN¹-(7-morpholinoquinoxalin-5-yl)-N⁴-(pyrimidin-2-yl)cyclohexane-1,4-diamine(Compound 18)

As shown in step 3-i of Scheme 3, to a solution of3-bromo-5-fluoro-benzene-1,2-diamine (compound 1001, 1.11 g, 5.41 mmol)in methanol (11 mL) was added oxaldehyde (1.57 mL of 40% w/v, 10.8mmol). The reaction mixture was stirred at room temperature undernitrogen. After 2 hours a yellow solid precipitated. The reactionmixture was diluted with water (20 mL), stirred an additional 5 minutes,filtered, and the collected solid dried under high vacuum to produce5-bromo-7-fluoroquinoxaline (compound 1002, 868 mg, 70.6% yield): ¹H-NMR(300 MHz, DMSO-d₆) δ 9.06 (s, 2H), 8.36 (dd, J=8.5, 2.7 Hz, 1H), 8.00(dd, J=9.2, 2.7 Hz, 1H); ESMS (M+H⁺)=227.14.

As shown in step 3-ii of Scheme 3, to a solution of5-bromo-7-fluoroquinoxaline (4.5 g, 19.8 mmol) in NMP (67.5 mL) wasadded morpholine (3.1 mL, 35.6 mmol). The reaction mixture was heated to140° C. and stirred for 15 hours. After cooling, the mixture was pouredinto water (200 mL), extracted with ethyl acetate (2×100 mL), dried overmagnesium sulfate, filtered, evaporated under reduced pressure, andpurified by medium pressure silica gel chromatography (10 to 80%EtOAc/hexanes gradient) to provide 4-(8-bromoquinoxalin-6-yl)morpholine(compound 1003, 3.86g, 66% yield) as a yellow solid: ¹H-NMR (400 MHz,DMSO-d₆) δ 8.82 (d, J=1.6 Hz, 1H), 8.73 (d, J=1.6 Hz, 1H), 8.12 (d,J=2.5 Hz, 1H), 7.27 (d, J=2.4 Hz, 1H), 3.87-3.69 (m, 4H), 3.44-3.34 (m,4H); ESMS (M+H⁺)=227.14.

As shown in step 3-iii of Scheme 3, a mixture of4-(8-bromoquinoxalin-6-yl)morpholine (1.57 g, 5.34 mmol),tert-butyl-N-(4-aminocyclohexyl)carbamate (1.37 g, 6.40 mmol),(rac)-BINAP (664 mg, 1.07 mmol), cesium carbonate (5.22 g, 16.0 mmol),and Pd₂(dba)₃ (489 mg, 0.534 mmol) in toluene (50 mL) was heated at 100°C. for 12 hours. After cooling, the mixture was diluted with ethylacetate (150 mL) and water (25 mL), then filtered through diatomaceousearth which was subsequently washed with ethyl acetate. The combinedorganics were washed with brine, dried over sodium sulfate, concentratedunder reduced pressure, and purified by medium pressure silica gelchromatography (0 to 60% EtOAc/hexanes gradient) to providetert-butyl(-4-((7-morpholinoquinoxalin-5-yl)amino)cyclohexyl)carbamate(compound 1004, 1.83g, 83.2% yield): ¹H-NMR (300 MHz, CDCl₃) δ 8.65 (d,J=2.0 Hz, 1H), 8.35 (d, J=2.0 Hz, 1H), 6.60 (d, J=2.4 Hz, 1H), 6.34 (d,J=2.4 Hz, 1H), 6.11 (d, J=7.8 Hz, 1H), 4.60 (s, 1H), 3.97-3.86 (m, 4H),3.67 (s, 2H), 3.41-3.25 (m, 4H), 1.85 (d, J=3.0 Hz, 5H), 1.74-1.57 (m,3H), 1.45 (s, 9H).

As shown in step 3-iv of Scheme 3, to a solution of tert-butyl(-4-((7-morpholinoquinoxalin-5-yl)amino)cyclohexyl)carbamate (900 mg,2.00 mmol) in dichloromethane (16 mL) was added trifluoroacetic acid (3mL, 38.9 mmol). The resulting black reaction mixture was stirred underan atmosphere of nitrogen at room temperature for 2 hours. Saturatedaqueous sodium bicarbonate (150 mL) was added slowly until the colorturned from black to orange. The mixture was extracted withdichloromethane (2×100 mL) and the combined organics washed with brine(50 mL), dried over sodium sulfate, and concentrated under reducedpressure toprovide-N¹-(7-morpholinoquinoxalin-5-yecyclohexane-1,4-diamine,trifluoroacetate (compound 1005): ¹H NMR (300 MHz, CDCl₃) δ 8.64 (d,J=1.9 Hz, 1H), 8.36 (d, J=1.9 Hz, 1H), 6.59 (d, J=2.3 Hz, 1H), 6.34 (d,J=2.3 Hz, 1H), 6.20 (d, J=7.9 Hz, 1H), 3.95-3.84 (m, 4H), 3.69 (s, 1H),3.41-3.25 (m, 4H), 2.93 (d, J=8.9 Hz, 1H), 2.09-1.87 (m, 2H), 1.90-1.68(m, 6H), 1.58 (dd, J=11.2, 8.7 Hz, 2H); ESMS (M+H⁺)=328.34. Thiscompound was used as is without further purification.

As shown in step 3-v of Scheme 3, to solution ofN¹-(7-morpholinoquinoxalin-5-yl)cyclohexane-1,4-diamine (25 mg, 0.07mmol) and diisopropylethylamine (18.0 mg, 24.3 μL, 0.14 mmol) indichloromethane (750 μL) was added ethyl chloroformate (11.4 mg, 10.0μL, 0.105 mmol). The reaction mixture was stirred for 12 hours, dilutedwith dichloromethane (10 mL), washed with saturated aqueous sodiumbicarbonate (5 mL), dried over sodium sulfate, and concentrated underreduced pressure. The resulting residue was purified by HPLC preparativechromatography using a 10-90% acetonitrile/water (0.1% TFA) gradient aseluant to provide ethyl(4-((7-morpholinoquinoxalin-5-yl)amino)cyclohexyl)carbamate (compound 6,14 mg, 50% yield): ¹H-NMR (300 MHz, CDCl₃) δ 8.65 (d, J=2.0 Hz, 1H),8.36 (d, J=2.0 Hz, 1H), 6.61 (d, J=2.4 Hz, 1H), 6.35 (d, J=2.4 Hz, 1H),6.10 (d, J=7.6 Hz, 1H), 4.72 (s, 1H), 4.12 (q, J=7.0 Hz, 2H), 3.96-3.82(m, 4H), 3.68 (s, 2H), 3.42-3.23 (m, 4H), 1.93-1.78 (m, 6H), 1.69 (dd,J=15.0, 6.3 Hz, 2H), 1.25 (t, J=7.1 Hz, 3H); ESMS (M+H⁺)=400.17.

As shown in step 3-vi of Scheme 3, A mixture ofN¹-(7-morpholinoquinoxalin-5-yl)cyclohexane-1,4-diamine (185 mg, 0.56mmol), 2-bromopyrimidine (93 mg, 0.58 mmol), and triethylamine (143 mg,197 μL, 1.41 mmol) in 1-methylpyrrolidin-2-one (3 mL) was heated to 130°C. and stirred for 15 hours. After cooling to room temperature, themixture was diluted with ethyl acetate (70 mL) and methyl tert-butylether (20 mL), washed with water (3×20 mL), washed with brine (15 mL),dried over sodium sulfate, concentrated under reduced pressure, andpurified by medium pressure silica gel chromatography (10 to 100%EtOAc/hexanes gradient) to provideN¹-(7-morpholinoquinoxalin-5-yl)-N⁴-(pyrimidin-2-yecyclohexane-1,4-diamine(compound 18, 102 mg, 45% yield): ¹H-NMR (300 MHz, CDCl₃) δ 8.65 (d,J=2.0 Hz, 1H), 8.37 (d, J=2.0 Hz, 1H), 8.27 (d, J=4.8 Hz, 2H), 6.60(d,J=2.4 Hz, 1H), 6.51 (t, J=4.8 Hz, 1H), 6.36 (d, J=2.4 Hz, 1H), 6.15 (d,J=7.8 Hz, 1H), 5.20 (d, J=7.7 Hz, 1H), 4.04 (d, J=7.9 Hz, 1H), 3.96-3.82(m, 4H), 3.70 (s, 1H), 3.39-3.24 (m, 4H), 1.94 (dd, J=13.7, 4.4 Hz, 6H),1.78 (dt, J=28.8, 16.1 Hz, 2H); ESMS (M+H⁺)=328.34.

EXAMPLE 4 Preparation of1-(4-((7-(8-oxa-3-azabicyclo[3.2.1]octan-3-yl)quinoxalin-5-yl)amino)cyclohexyl)-3-ethylurea(Compound 22)\

As shown in step 4-i of Scheme 4, to a solution of5-bromo-7-fluoroquinoxaline (compound 1002, 150 mg, 0.66 mmol) in NMP(2.3 mL) was added 8-oxa-3-azabicyclo[3.2.1]octane (178 mg, 1.2 mmol) atRT. The reaction mixture was sealed in a microwave vial and heated at180° C. for 20 minutes. Afte cooling to RT and pouring into water, theaqueous phase was extracted with EtOAc (3×). The combined extracts weredried over MgSO₄, filtered, concentrated under reduced pressure, andpurified by medium pressure silica gel chromatography (0 to 100%EtOAc/hexanes gradient) to provide3-(8-bromoquinoxalin-6-yl)-8-oxa-3-azabicyclo[3.2.1]octane (compound1006, 87 mg, 41% yield) as a dark orange oil: ESMS (M+H⁺)=320.07.

As shown in step 4-ii of Scheme 4, a degassed solution of3-(8-bromoquinoxalin-6-yl)-8-oxa-3-azabicyclo[3.2.1]octane (261 mg,0.815 mmol), tert-butyl N-(4-aminocyclohexyl)carbamate (210 mg, 0.98mmol), rac-BINAP (102 mg, 0.163 mmol), Cs₂CO₃ (797 mg, 2.45mmo1), andPd₂(dba)₃ (75 mg, 0.0815 mmol) in toluene (10.5 mL) was heated at 100°C. (oil bath temp) in a sealed microwave tube for 15 hours. Aftercooling, the mixture was applied directly to a chromatography column andpurified by medium pressure silica gel chromatography (0 to 100%EtOAc/hexanes gradient) to afford tert-butyl(4-((7-(8-oxa-3-azabicyclo[3.2.1]octan-3-yl)quinoxalin-5-yl)amino)cyclohexyl)carbamate(compound 1007, 141 mg, 36% yield) as a white solid: ¹H-NMR (400 MHz,CDCl₃) δ 8.49 (s, 1H), 8.23 (d, J=1.5 Hz, 1H), 6.48 (s, 1H), 6.18 (d,J=1.9 Hz, 1H), 6.06 (s, 1H), 4.52 (s, 1H), 4.47 (s, 2H), 3.60 (s, 2H),3.45 (d, J=11.6 Hz, 2H), 3.14-3.12 (m, 2H), 1.96-1.84 (m, 4H), 1.79 (s,5H), 1.54 (s, 3H) and 1.38 (s, 9H) ppm; ESMS (M+H⁺)=453.96.

As shown in step 4-iii of Scheme 4, to a solution of compound 1007 (141mg, 0.295 mmol) in CH₂Cl₂ (2.5 mL) was added TFA (656 mg, 443 μL, 5.75mmol) at RT. The resulting black solution was stirred for 2 hours andthen the reaction was quenched by the addition of saturated NaHCO₃ untilthe black color gradually turned into an orange color. The reactionmixture was extracted with CH₂Cl₂ (3×) and the combined organic extractswere dried over Na₂SO₄ and evaporated to dryness to provideN¹-(7-(8-oxa-3-azabicyclo[3.2.1]octan-3-yl)quinoxalin-5-yl)cyclohexane-1,4-diamine,trifluoroacetate (compound 1008): ESMS (M+H⁺)=354.20. This material wasused in subsequent reactions without any further purification.

As shown in step 4-iv of Scheme 4, to a solution of compound 1008 (45mg, 0.071 mmol) and DIEA (36.5 mg, 49.0 μL, 0.28 mmol) in CH₂Cl₂ (1.4mL) was added ethyl isocyanate (20 mg, 0.28 mmol) at RT. The solutionwas stirred at this temperature for 15 hours and then applied directlyto a chromatography column and purified by medium pressure silica gelchromatograph (0 to 100% EtOAc/hexanes gradient) to afford1-(4-((7-(8-oxa-3-azabicyclo[3.2.1]octan-3-yl)quinoxalin-5-yl)amino)cyclohexyl)-3-ethylurea(compound 22, 8 mg, 27% yield) as a white solid: ¹H-NMR (400 MHz, CDCl₃)δ 8.54 (s, 1H), 8.22 (s, 1H), 6.43 (s, 1H), 6.19 (s, 1H), 6.02 (s, 1H),4.47 (s, 2H), 4.38 (d, J=5.2 Hz, 1H), 4.28 (s, 1H), 3.74 (s, 1H), 3.60(s, 1H), 3.42 (s, 4H), 3.14-3.09 (m, 4H), 2.05-1.87 (m, 3H), 1.79 (s,3H), 1.55 (d, J=7.1 Hz, 2H) and 1.21-1.05 (m, 5H) ppm; ESMS(M+H⁺)=425.35.

EXAMPLE 5 Preparation ofN¹-(6-morpholinobenzo[c][1,2,5]oxadiazol-4-yl)-N⁴-(pyrimidin-2-yl)cyclohexane-1,4-diamine(Compound 23)

As shown in step 5-i of Scheme 5, a mixture of tert-butyl((cis)-4-aminocyclohexyl)carbamate (compound 1009, 490 mg, 2.3 mmol),2-chloropyrimidine (262 mg, 2.3 mmol) and TEA (463 mg, 637μL, 4.6 mmol)in DMF (10 mL) was subjected to microwave irradiation for 20 minutes at150° C. The reaction mixture was diluted with EtOAc, washed with H₂O,dried over Na₂SO₄, concentrated under reduced pressure, and purified bymedium pressure silica gel chromatography (0 to 50% EtOAc/hexanesgradient) to provide tert-butyl((cis)-4-(pyrimidin-2-ylamino)cyclohexyl)carbamate (compound 1010) as awhite solid: ¹H-NMR (300 MHz, CDCl₃) δ 8.28 (d, J=4.8 Hz, 2H), 6.53 (t,J=4.8 Hz, 1H), 5.12 (s, 1H), 4.56 (s, 1H), 3.99 (dq, J=7.0, 3.5 Hz, 1H),3.65 (s, 1H), 1.83 (tq, J=10.2, 3.6 Hz, 5H), 1.66 (s, 8H), 8.13-7.91 (m,3H), 1.47 (s, 9H).

As shown in step 5-ii of Scheme 5, HCl (3 mL, 4M in THF, 12 mmol) wasadded to compound 1010. The mixture was stirred for 30 min andconcentrated under reduced pressure to produce(cis)-N¹-(pyrimidin-2-yl)cyclohexane-1,4-diamine hydrochloride (compound1011). This material was used in subsequent reactions as is withoutfurther purification.

As shown in step 5-iii of Scheme 5, a mixture of4-bromo-6-morpholinobenzo[c][1,2,5]oxadiazole (compound 1012, 147 mg,0.5 mmol), (cis)-N¹-(pyrimidin-2-yl)cyclohexane-1,4-diaminehydrochloride (120 mg, 0.6 mmol), (rac)-BINAP (32 mg, 0.05 mmol) ,Pd₂(dba)₃ (24 mg, 0.026 mmol), and cesium carbonate (506 mg, 1.55 mmol)in toluene (5 mL) was flushed with nitrogen gas and stirred overnight at90° C. under an atmosphere of nitrogen. The mixture was filtered thougha layer of diatomaceous earth, concentrated under reduced pressure, andpurified by medium pressure silica gel chromatography (0 to 80%EtOAc/hexanes gradient) to provide(cis)-N¹-(6-morpholinobenzo[c][1,2,5]oxadiazol-4-yl)-N⁴-(pyrimidin-2-yl)cyclohexane-1,4-diamine(compound 23) as an orange solid: ¹H-NMR (300 MHz, CDCl₃) δ 8.20 (d,J=4.9 Hz, 2H), 6.46 (t, J=4.8 Hz, 1H), 6.05 (d, J=1.6 Hz, 1H), 5.82 (s,1H), 5.24 (s, 1H), 4.82 (d, J=7.0 Hz, 1H), 3.98 (s, 1H), 3.85-3.72 (m,4H), 3.60 (s, 1H), 3.23-3.06 (m, 4H), 1.95-1.62 (m, 8H).

EXAMPLE 6 Preparation of5-methoxy-N-((cis)-4-((7-morpholinoquinoxalin-5-yl)oxy)cyclohexyl)pyrimidin-2-amine(Compound 134)

As shown in step 6-i of Scheme 6, to a mixture of5-bromo-2-fluoro-pyrimidine (1 g, 5.651 mmol) in iPrOH (10 mL) was addedTEA (1.143 g, 1.574 mL, 11.30 mmol) and trans-4-aminocyclohexan-1-ol(650.8 mg, 5.651 mmol). The mixture was microwaved for 20min at 150° C.,concentrated under reduced pressure, diluted with EtOAc , washed withwater, and dried over Na₂SO_(4.) After removal of the volatiles underreduced pressure, the residue was purified by medium pressure silica gelchromatography (0-80% EtOAc/hexanes gradient) to provide(trans)-4-((5-bromopyrimidin-2-yl)amino)cyclohexanol (compound 1013, 1.2g): ¹H-NMR (300 MHz, CDCl₃) δ 8.28 (s, 2H), 5.03 (d, J=8.1 Hz, 1H),3.91-3.49 (m, 2H), 2.31-1.90 (m, 4H), 1.56-1.19 (m, 4H).

As shown in step 6-ii of Scheme 6, to compound 1013 (1.2 g, 4.41 mmol)in DCM (20 mL) was added TEA (1.134 g, 1.84 mL, 13.2 mmol) and MsCl (505mg, 341 μL, 4.41 mmol). The reaction mixture was stirred for 1 hour,concentrated under reduced pressure, and purified by medium pressuresilica gel chromatography (0 to 80% EtOAc/hexanes gradient) to providetrans-4-((5-bromopyrimidin-2-yl)amino)cyclohexyl methanesulfonate(compound 1014): ¹H-NMR (300 MHz, CDCl₃) δ 8.29 (s, 2H), 5.03 (d, J=7.8Hz, 1H), 4.70 (tt, J=10.6, 3.9 Hz, 1H), 3.80 (dtt, J=11.2, 7.6, 3.7 Hz,1H), 3.04 (s, 3H), 2.30-2.12 (m, 4H), 1.93-1.69 (m, 2H), 1.51-1.33 (m,2H).

As shown in step 6-iii of Scheme 6, to a solution of2-amino-3-nitrophenol (5.00 g, 32.4 mmol) in dioxane (50 mL) was addedbromine (6.22 g, 2.01 mL, 38.9 mmol). The mixture was stirred for 2hours and a precipitate formed, which was collected and washed withdioxane and ether. The resulting yellow solid treated with a saturatedNaHCO₃ solution, which was extracted with EtOAc (3×). The combinedorganics were dried over Na₂SO_(4,) filtered, and concentrated underreduced pressure to yield 2-amino-5-bromo-3-nitrophenol (compound 1015)as a brown solid. This material was carried on as is in subsequentreactions without futher purification.

As shown in step 6-iv of Scheme 6, to a solution of2-amino-5-bromo-3-nitrophenol (7.5 g, 31.8 mmol) in ethyl acetate (60mL) was added Raney nickel™ (1.90 g, 214 μL, 32.4 mmol) and the reactionmixture was shaken for 2 hours under an atmosphere of H₂ at 30 p.s.i.After filtering and drying over Na₂SO_(4,) the mixture was concentratedunder reduced pressure to provide 2,3-diamino-5-bromophenol (compound1016), which was used as is in subsequent reactions without futherpurification.

As shown in step 6-v of Scheme 6, 2,3-diamino-5-bromophenol (6.0 g, 29.5mmol) was dissolved in methanol and to this solution was added glyoxal(3.77 g, 2.98 mL, 64.9 mmol) and stirred overnight. The reaction mixturewas concentrated under reduced pressure to a minimum volume and theresulting tan solid collected by filtration and dried under high vacuumto produce 7-bromoquinoxalin-5-ol (compound 1017), which was used as isin subsequent reactions without futher purification.

As shown in step 6-vi of Scheme 6, a solution of 7-bromoquinoxalin-5-ol(2.0 g, 8.89 mmol) in DCM (20 mL) was added imidazole (1.82 g, 26.7mmol) and tert-butyldimethylsilyl chloride (1.34 g, 1.65 mL, 8.89 mmol).The reaction mixture was stirred overnight at RT, concentrated underreduced pressure, and purified by medium pressure silica gelchromatography (0 to 20% EtOAc/hexanes gradient) to provide7-bromo-5-((tert-butyldimethylsilyl)oxy)quinoxaline (compound 1018) as acolorless oil: ¹H-NMR (300 MHz, CDCL₃) δ 8.69 (q, J=1.8 Hz, 2H), 7.80(d, J=2.1 Hz, 1H), 7.22 (d, J=2.1 Hz, 1H), 0.96 (s, 9H), 0.81 (s, 7H).

As shown in step 6-vii of Scheme 6, a mixture of7-bromo-5-((tert-butyldimethylsilyl)oxy)quinoxaline (700 mg, 2.06 mmol),morpholine (270 mg, 270 L, 3.09 mmol), Pd₂(dba)₃ (94.50 mg, 0.1032mmol), (rac)-BINAP (129 mg, 0.206 mmol), cesium carbonate (2.02 g, 6.19mmol) in toluene (7 mL) was flushed with nitrogen for 10 minutes. Themixture was then heated overnight at 100° C. After cooling, the reactionmixture was diluted with EtOAc, filtered through a layer of diatomaceousearth, concentrated under reduced pressure, and purified by mediumpressure silica gel chromatography (0 to 30% EtOAc/hexanes gradient) toprovide 7-morpholinoquinoxalin-5-ol. This compound (450 mg, 1.3 mmol)was dissolved in THF (20 mL) and tetra-n-butylammonium fluoride (539 mg,2.06 mmol) was added. The reaction mixture was stirred for 0.5 hour,concentrated under reduced pressure, and purified by medium pressuresilica gel chromatography (0 to 100% EtOAc/hexanes gradient) to provide7-morpholinoquinoxalin-5-ol (compound 1019) as a yellow solid: ¹H-NMR(300 MHz, CDCl₃) δ 8.75 (d, J=2.0 Hz, 1H), 8.46 (d, J=2.0 Hz, 1H), 7.70(d, J=41.8 Hz, 1H), 7.01 (d, J=2.6 Hz, 1H), 6.89 (d, J=2.5 Hz, 1H),4.12-3.78 (m, 4H), 3.51-3.24 (m, 4H).

As shown in step 6-viii of Scheme 6, a solution of7-morpholinoquinoxalin-5-ol (100 mg, 0.432 mmol),(trans)-4-((5-bromopyrimidin-2-yl)amino)cyclohexyl methanesulfonate(compound 1014, 303 mg, 0.865 mmol), and CsCO₃ (282 mg, 0.865 mmol) indioxane (1.0 mL was stirred for 16 hours at 105° C. After cooling, thereaction mixture was diluted with EtOAc, filtered through diatomaceousearth, concentrated under reduced pressure, and purified by mediumpressure silica gel chromatography (0 to 5% MeOH/DCM gradient) toproduce5-bromo-N-((cis)-4-((7-morpholinoquinoxalin-5-yl)oxy)cyclohexyl)pyrimidin-2-amine(compound 1020, 110 mg) as a yellow foam: ¹H-NMR (400 MHz, CDCl₃) δ 8.70(d, J=2.0 Hz, 1H), 8.64 (d, J=1.9 Hz, 1H), 8.29 (s, 2H), 6.98 (d, J=2.5Hz, 1H), 6.92 (d, J=2.5 Hz, 1H), 5.29 (d, J=8.3 Hz, 1H), 4.81 (s, 1H),4.04-3.84 (m, 4H), 3.42-3.31 (m, 4H), 2.22 (s, 2H), 1.92 (d, J=4.9 Hz,6H).

As shown in step 6-ix of Scheme 6, to a mixture5-bromo-N-((cis)-4-((7-morpholinoquinoxalin-5-yl)oxy)cyclohexyl)pyrimidin-2-amine(75 mg, 0.155 mmol), cesium carbonate (101 mg, 0.309 mmol) ,allylpalladium(II) chloride dimer (0.28 mg, 0.0015 mmol) , RockPhos(2.17 mg, 0.0046 mmol) and MeOH (9.9 mg, 12.5 μL, 0.31 mmol) in toluene(2 mL) was flushed with nitrogen gas and heated to 100° C. for 18 hours.The reaction mixture was iluted with EtOAc, filtered though a layer ofdiatomaceous earth, and concentrated under reduced pressure.Purification by medium pressure silica gel chromatography (0-8% MeOH/DCMgradient) yielded5-methoxy-N-((cis)-4-((7-morpholinoquinoxalin-5-yl)oxy)cyclohexyl)pyrimidin-2-amine(compound 134, 43 mg): ¹H-NMR (300 MHz, CDCl₃) δ 8.70 (d, J=1.9 Hz, 1H),8.63 (d, J=1.9 Hz, 1H), 8.07 (s, 2H), 6.96 (d, J=2.5 Hz, 1H), 6.92 (d,J=2.5 Hz, 1H), 5.01 (d, J=8.1 Hz, 1H), 4.80 (q, J=5.6, 4.2 Hz, 1H),4.03-3.87 (m, 5H), 3.80 (s, 3H), 3.42-3.27 (m, 4H), 2.29-2.10 (m, 2H),1.99-1.82 (m, 6H).

EXAMPLE 7 Preparation of4-(8-(((trans)-4-(pyrimidin-2-yloxy)cyclohexyl)oxy)quinoxalin-6-yl)morpholine(Compound 34) and4-(8-(((cis)-4-(pyrimidin-2-yloxy)cyclohexyl)oxy)-quinoxalin-6-yl)morpholine(Compound 42)

As shown in step 7-i of Scheme 7, to a solution of1,4-dioxaspiro[4.5]decan-8-ol (compound 1021, 1.0 g, 6.32 mmol) in DMF(10 mL) was added NaH (370 mg, 9.25 mmol). The reaction mixture wasstirred for 20 minutes before the addition of 2-chloropyrimidine (869mg, 7.59 mmol). The mixture was stirred for 30 minute at RT and thenheated to 100° C. for 9 hours. After cooling, the mixture was dilutedwith EtOAc, washed with H₂O, dried over Na₂SO_(4,) concentrated underreduced pressure, and purified by medium pressure silica gelchromatography (0-40% EtOAc/hexanes) to produce2-(1,4-dioxaspiro[4.5]decan-8-yloxy)pyrimidine (compound 1022) as acolorless oil: ¹H NMR (300 MHz, Chloroform-d) δ 8.52 (d, J=4.8 Hz, 2H),6.92 (t, J=4.8 Hz, 1H), 5.15 (ddd, J=10.7, 6.5, 4.2 Hz, 1H), 4.05-3.87(m, 4H), 2.14-1.85 (m, 6H), 1.79-1.65 (m, 2H); ESMS (M+H⁺)=237.12.

As shown in step 7-ii of Scheme 7, to2-(1,4-dioxaspiro[4.5]decan-8-yloxy)pyrimidine (620 mg, 2.624 mmol) wasadded HCl (4.0 mL of 6 M, 8.86 mmol) and the reaction mixture wasstirred for 2 hours. The pH of the mixture was neutralized with withsat. NaHCO₃(aq) and the mixture was concentrated under reduced pressureas a methanol azeotrope. To the residue was added DCM (30 mL) to producea precipitate, followed by stirring for an additional 20 minutes. Thesolids were filtered off and the mother liquor was concentrated underreduced pressure. The resulting residue was dissolved in methanol andsodium borohydride (151 mg, 3.99 mmol) was added as a solid. The mixturewas stirred for 1 hour and the reaction quenched with HCl (6M, 0.70 mL).Stirring was continued until gas evolution ceased. The pH of the mixturewas adjusted to about 8 with 1N sodium hydroxide and extracted withEtOAc (20 mL). The organics were dried over sodium sulfate andconcentrated under reduced pressure to produce4-(pyrimidin-2-yloxy)cyclohexanol (compound 1023, 248 mg, 64% yield) asa mixture of (cis)- and (trans)-isomers. A 12 mg aliquot of the samplewas purified via HPLC preperative reversed-phase chromatography (10-90%CH₃CN/water gradient containing 0.1% TFA) to separate the isomers:(trans)-4-pyrimidin-2-yloxycyclohexanol-¹H NMR (300 MHz, Chloroform-d) δ8.54 (d, J=4.8 Hz, 2H), 6.95 (t, J=4.8 Hz, 1H), 5.05 (tt, J=9.4, 4.0 Hz,1H), 3.91-3.75 (m, 1H), 2.26-1.99 (m, 4H), 1.76-1.41 (m, 4H); ESMS(M+H⁺)=195.07, (cis)-4-pyrimidin-2-yloxycyclohexanol NMR (300 MHz,Chloroform-d) δ 8.62 (d, J=4.9 Hz, 2H), 7.04 (t, J=4.9 Hz, 1H), 5.21(tt, J=5.3, 2.6 Hz, 1H), 4.56 (s, 1H), 3.85 (p, J=5.9 Hz, 1H), 2.17-2.02(m, 2H), 1.88-1.67 (m, 6H); ESMS (M+H⁺)=195.07. The remaining materialwas used in subsequent reactions as the cis/trans mixture.

As shown in step 7-iii of Scheme 7, to a solution of a cis/trans mixtureof 4-pyrimidin-2-yloxycyclohexanol (244 mg, 1.256 mmol) andtriethylamine (350 μL, 2.51 mmol) in dichloromethane (5 mL) was addedmethane sulfonyl chloride (145 μL, 1.87 mmol). The reaction mixture wasstirred for 2 hours, concentrated under reduced pressure, and purifiedby medium pressure silica gel chromatography (0-20%EtOAc/dichloromethane gradient) to provide4-pyrimidin-2-yloxycyclohexyl) methanesulfonate (compound 1024, 239 mg,70% yield) as a mixture of cis/trans isomers: ¹H NMR (300 MHz,Chloroform-d) δ 8.51 (d, J=4.8 Hz, 2H), 6.93 (t, J=4.8 Hz, 1H), 5.13(dq, J=9.9, 3.0 Hz, 1H), 4.87 (p, J=3.8 Hz, 1H), 3.04 (d, J=2.4 Hz, 3H),2.28-1.99 (m, 4H), 1.99-1.74 (m, 4H); ESMS (M+H⁺)=273.52.

As shown in step 7-iv of Scheme 7, a mixture of(4-pyrimidin-2-yloxycyclohexyl) methanesulfonate (105 mg, 0.386 mmol),7-morpholinoquinoxalin-5-ol (178.3 mg, 0.7712 mmol), and Cs2CO3 (125.6mg, 0.3856 mmol) in dioxane (1.5 mL) was sealed in a 5 mL microwave tubeand heated to 110° C. for 14 hours using an oil bath. The reactionmixture was cooled to room temperature, diluted with EtOAc, and filteredthrough diatomaceous earth which was subsequently washed with ethylacetate. The filtrate was concentrated under reduced pressure and theresidue purified via preparative reversed-phase HPLC (10-90% CH₃CN/watergradient containing 0.1% TFA). Fractions containing a mixture of cis andtrans isomers were further purified via SFC using a chiral OJ column andeluting with 40% MeOH in CO₂ to provide 21 mg of4-(8-(((trans)-4-(pyrimidin-2-yloxy)cyclohexyl)oxy)quinoxalin-6-yl)morpholine(compound 34): ¹H NMR (300 MHz, Chloroform-d) δ 8.69 (dd, J=3.4, 1.9 Hz,1H), 8.62 (dd, J=3.6, 1.9 Hz, 1H), 8.51 (dd, J=4.8, 2.2 Hz, 2H),7.01-6.83 (m, 3H), 5.18 (tt, J=7.0, 3.4 Hz, 1H), 4.79 (tt, J=6.9, 3.1Hz, 1H), 4.00-3.85 (m, 4H), 3.34 (dq, J=4.8, 2.6 Hz, 4H), 2.44-2.16 (m,4H), 1.92 (tdd, J=16.4, 7.7, 2.8 Hz, 4H); ESMS (M+H⁺)=408.56, and 22 mgof4-(8-(((cis)-4-(pyrimidin-2-yloxy)cyclohexyl)oxy)-quinoxalin-6-yl)morpholine(compound 42): ¹H NMR (300 MHz, Chloroform-d) δ 8.70 (d, J=1.9 Hz, 1H),8.63 (d, J=1.9 Hz, 1H), 8.52 (d, J=4.8 Hz, 2H), 7.01-6.87 (m, 3H), 5.17(ddt, J=8.7, 6.7, 3.4 Hz, 1H), 4.76-4.58 (m, 1H), 4.00-3.87 (m, 4H),3.40-3.27 (m, 4H), 2.43-2.22 (m, 4H), 2.05-1.87 (m, 2H), 1.86-1.71 (m,2H); ESMS (M+H⁺)=408.56.

EXAMPLE 8N-[(cis)-4-[7-(3,6-dihydro-2H-pyran-4-yl)quinoxalin-5-yl]oxycyclohexyl]pyrimidin-2-amine(Compound 36)

As shown in step 8-i of Scheme 8, to a mixture of 7-bromoquinoxalin-5-ol(compound 1018, 200 mg, 0.89 mmol) and cesium carbonate (579 mg, 1.78mmol) in NMP (4.0 mL) was added(trans)-4-(pyrimidin-2-ylamino)cyclohexyl methanesulfonate (compound1014, 241.1 mg, 0.8887 mmol). The mixture was stirred for 18 hours at90° C., at which time an additional 0.5 eq of compound 1014 (241 mg,0.89 mmol) was added. After stirring at 90° C. for an additional 6hours, the reaction mixture was diluted with EtOAc, washed with H₂O,dried over Na₂SO_(4,) concentrated, and purified by medium pressuresilica gel chromatography (0-5% MeOH/DCM) to provideN-((cis)-4-((7-bromoquinoxalin-5-yl)oxy)cyclohexyl)pyrimidin-2-amine(compound 1025): ¹H-NMR (300 MHz, CDCl₃) δ 9.01-8.77 (m, 2H), 8.29 (d,J=4.8 Hz, 2H), 7.89 (d, J=1.9 Hz, 1H), 7.25 (d, J=2.0 Hz, 1H), 6.53 (t,J=4.8 Hz, 1H), 5.43-5.22 (m, 1H), 4.79 (td, J=5.2, 2.5 Hz, 1H),4.18-3.95 (m, 1H), 3.51 (s, 1H), 2.22 (td, J=10.2, 9.6, 5.4 Hz, 2H),2.09-1.86 (m, 6H).

As shown in step 8-ii of Scheme 8, a mixture ofN-((cis)-4-((7-bromoquinoxalin-5-yl)oxy)cyclohexyl)pyrimidin-2-amine(compound 1025, 52 mg, 0.1299 mmol) , Pd(dppf)Cl₂ (10.61 mg, 0.01299mmol),2-(3,6-dihydro-2H-pyran-4-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane(compound 1026, 27.3 mg, 0.13 mmol), Na₂CO₃ (195 μL of 2M (aq) solution,0.39 mmol) in DMF (1 mL) was flushed with nitrogen gas for 10 minutes.The mixture was subjected to microwave radiation for 20 min at 150° C.After cooling, the mixture was diluted with EtOAc , washed with H₂O,dried over Na₂SO_(4,) concentrated under reduced pressure and purifiedby medium pressure silica gel chromatography (0-5% MeOH/DCM) to provideN-[(cis)-4-[7-(3,6-dihydro-2H-pyran-4-yl)quinoxalin-5-yl]oxycyclohexyl]pyrimidin-2-amine(compound 36) as an off-white solid: ¹H-NMR (300 MHz, CDCl₃) δ 8.94-8.76(m, 2H), 8.29 (d, J=4.8 Hz, 2H), 7.67 (d, J=1.7 Hz, 1H), 6.53 (t, J=4.8Hz, 1H), 6.37 (tt, J=3.1, 1.5 Hz, 1H), 5.30 (d, J=7.9 Hz, 1H), 4.87 (dt,J=7.5, 3.6 Hz, 1H), 4.43 (q, J=2.8 Hz, 2H), 4.02 (t, J=5.5 Hz, 3H), 2.68(dqd, J=6.0, 3.4, 3.0, 1.8 Hz, 2H), 2.35-2.11 (m, 2H), 2.07-1.84 (m,6H); ESMS (M+H⁺)=404.2.

EXAMPLE 9N-((cis)-4-((7-morpholinoquinoxalin-5-yl)oxy)cyclohexyl)pyrimidin-2-amine(Compound 28)

As shown in step 9-i of Scheme 9, 7-bromoquinoxalin-5-ol (compound 1017,5.4 g, 24.0 mmol), 2-((trans)-4-hydroxycyclohexyl)isoindoline-1,3-dione(5.607 g, 22.86 mmol), and triphenylphosphine (8.994 g, 7.945 mL, 34.29mmol) were dissolved in anhydrous THF and the flask was cooled in an icebath. DIAD (6.93 g, 6.64 mL, 34.3 mmol) was added dropwise and thereaction was stirred at 0° C. for 5 minutes, then warmed to roomtemperature and stirred for 18 hours. The reaction mixture wasconcentrated under reduced pressure, the residue was treated with Et₂Oand stirred for 0.5 hour at RT, the precipitates filtered off, thefiltrate concentrated under reduced pressure, and the residue purifiedby medium pressure silica gel chromatography (0-50% EtOAc/hexanesgradient) to produce2-[(cis)-4-(7-bromoquinoxalin-5-yl)oxycyclohexyl]isoindoline-1,3-dione(compound 1028, 6.2 g, 60% yield): ¹H-NMR (300 MHz, CDCl₃) δ 8.95 (d,J=1.8 Hz, 1H), 8.86 (d, J=1.8 Hz, 1H), 7.91 (d, J=2.0 Hz, 1H), 7.88-7.80(m, 2H), 7.77-7.68 (m, 2H), 7.31 (d, J=2.0 Hz, 1H), 4.96 (t, J=2.9 Hz,1H), 4.29 (tt, J=12.5, 3.8 Hz, 1H), 2.88 (qd, J=12.9, 3.6 Hz, 2H),2.54-2.32 (m, 2H), 1.94-1.61 (m, 4H).

As shown in step 9-ii of Scheme 9, In a round bottom flask fitted with acondenser, a mixture of2-[4-(7-bromoquinoxalin-5-yl)oxycyclohexyl]isoindoline-1,3-dione (6.2 g,12.34 mmol) , morpholine (1.61 g, 1.62 mL, 18.5 mmol) , and Cs₂CO₃(12.06 g, 37.0 mmol) in anhydrous toluene (73 mL) was treated withrac-BINAP (768.4 mg, 1.234 mmol) and Pd₂(dba)₃ (565 mg, 0.617 mmol). Thereaction mixture was heated at 110° C. for 18 hours. After cooling toroom temperature, the mixture was filtered through diatomaceous earthand concentrated under reduced pressure. The residue was triturated withEt₂O and the solids collected by filtration and washed with Et₂O toproduce2-((cis)-4-((7-morpholinoquinoxalin-5-yl)oxy)cyclohexyl)isoindoline-1,3-dione(compound 1029, 4.2 g) as yellow solid. The filterate was concentratedunder reduced pressure and purified by medium pressure silica gelchromatography (0-100% EtOAc/hexanes gradient) to produce an additional300 mg of compound 1029: ¹H-NMR (300 MHz, CDCl₃) δ 8.76-8.63 (m, 2H),7.85 (dd, J=5.4, 3.1 Hz, 2H), 7.79-7.60 (m, 2H), 7.09 (d, J=2.6 Hz, 1H),6.99 (d, J=2.5 Hz, 1H), 5.06 (t, J=2.8 Hz, 1H), 4.27 (tt, J=12.3, 3.8Hz, 1H), 4.02-3.85 (m, 4H), 3.49-3.27 (m, 4H), 3.03-2.75 (m, 2H), 2.37(d, J=14.0 Hz, 2H), 1.83-1.56 (m, 4H).

As shown in step 9-iii of Scheme 9, to a suspension of2-[(cis)-4-(7-morpholinoquinoxalin-5-yl)oxycyclohexyl]isoindoline-1,3-dione(2.3 g, 5.02 mmol) in MeOH (25 mL) was added hydrazine (321 mg, 315 μL,10.0 mmol) and the reaction mixture stirred for 18 hours at RT, overwhich time the initial suspension became homogenenous followed by theappearance of a precipitate. Et₂O (30 mL) was added and the reactionmixture stirred an additional 30 minutes. The precipitates were filteredoff, the filtrate concentrated under reduced pressure, the residuetreated with DCM (30 mL), and any remaining solids removed byfiltration. The filtrate was concentrated under reduced pressure toprovide (cis)-4-((7-morpholinoquinoxalin-5-yl)oxy)cyclohexanamine(compound 1030), which was used as is in subsequent reactions: ¹H-NMR(300 MHz, CDCl₃) δ 8.69 (d, J=1.9 Hz, 1H), 8.62 (d, J=1.9 Hz, 1H), 6.95(d, J=2.5 Hz, 1H), 6.90 (d, J=2.5 Hz, 1H), 5.00-4.67 (m, 3H), 4.03-3.81(m, 4H), 3.49 (s, 1H), 3.43-3.25 (m, 4H), 2.88 (q, J=6.2 Hz, 2H),2.36-1.96 (m, 6H).

As shown in step 9-iv of Scheme 9, to a solution so (cis)4-(7-morpholinoquinoxalin-5-yl)oxycyclohexanamine (415 mg, 1.264 mmol)and 2-methylsulfonylpyrimidine (400 mg, 2.53 mmol) was added DIEA (490mg, 661 μL, 3.79 mmol) and the reaction mixture was sealed in a vesseland heated to 100° C. for 16 hours. After this time, the volatiles wereremoved under a stream of nitrogen gas and the crude residue dissolvedin minimal amount of DCM. Purification by medium pressure silica gelchromatography (0-10% MeOH/DCM, 1% Et₃N producedN-((cis)-4-((7-morpholinoquinoxalin-5-yl)oxy)cyclohexyl)pyrimidin-2-aminecontaining triethylamine hydrochloride as an impurity. Dissolved productin DCM and stirred with a silica-supported amine (Silabond amine® 40-63μm). The scavenger mixture was filtered, concentrated under reducedpressure, and dried under high vacuum to provideN-((cis)-4-((7-morpholinoquinoxalin-5-yl)oxy)cyclohexyl)pyrimidin-2-amine(Compound 28, 435 mg): ¹H-NMR (400 MHz, CDCl₃) δ 8.68 (d, J=1.9 Hz, 1H),8.61 (d, J=1.9 Hz, 1H), 8.27 (s, 1H), 8.26 (s, 1H), 6.94 (d, J=2.4 Hz,1H), 6.90 (d, J=2.4 Hz, 1H), 6.50 (t, J=4.8 Hz, 1H), 4.78 (s, 1H),4.08-3.97 (m, 1H), 3.94-3.86 (m, 4H), 3.37-3.28 (m, 4H), 2.20 (d, J=9.1Hz, 2H), 1.95-1.85 (m, 6H).

EXAMPLE 10 Preparation ofN-[4-[7-(6-oxa-3-azabicyclo[3.1.1]heptan-3-yl)quinoxalin-5-yl]oxycyclohexyl]pyrimidin-2-amine(Compound 291)

To a mixture of 7-bromoquinoxalin-5-ol (47.53 g, 211.2 mmol),2-(4-hydroxycyclohexyl)isoindoline-1,3-dione (52.41 g, 213.7 mmol), andPPh3 (87.31 g, 332.9 mmol) in THF (740 mL) at 21° C. was addedtert-butyl (NZ)-N-tert-butoxycarbonyliminocarbamate (DTBAD) (79.51 g,328.0 mmol) in portions over 40 min so as to maintain the temperaturebelow 30° C. and the resultant reaction mixture was stirred at roomtemperature for a further 20 h.

The reaction was evaporated in vacuo. The residual reddish-brown viscousoil was dissolved in CH2C12 and filtered through a plug of silica in aglass column using applied air pressure (plug was made with 1 L of drysilica suspended in CH2Cl2). The plug was eluted with CH2Cl2, thefractions were combined and evaporated in vacuo to afford a red-brownviscous oil/foam, that was then dissolved in 700 mL of MeOH beforeprecipitating. The mixture was stirred at room temperature for 1 h,filtered, washed with cold MeOH (500 mL) and Et2O (100 mL), then driedin vacuo to yield a tan solid that was suspended in 300 mL MeOH andbrought to reflux for 10 min The suspension was cooled to roomtemperature and filtered, washed with a further MeOH and Et2O (4:1), anddried in vacuo to provide2-[4-(7-bromoquinoxalin-5-yl)oxycyclohexyl]isoindoline-1,3-dione (58.43g, 126.6 mmol, 59.94%). 1H NMR (400 MHz, CDCl₃) δ 8.96 (d, J=1.8 Hz,1H), 8.86 (d, J=1.8 Hz, 1H), 7.91 (d, J=1.9 Hz, 1H), 7.89-7.82 (m, 2H),7.78-7.67 (m, 2H), 7.30 (d, J=1.9 Hz, 1H), 4.95 (s, 1H), 4.29 (tt,J=12.5, 3.7 Hz, 1H), 2.87 (qd, J=13.1, 3.5 Hz, 2H), 2.44 (d, J=15.2 Hz,2H), 1.80 (t, J=14.1 Hz, 2H), 1.67 (d, 2H). ESI-MS m/z calc. 451.05316,found 452.19 (M+1)+; Retention time: 0.92 minutes.

A mixture of2-[4-(7-bromoquinoxalin-5-yl)oxycyclohexyl]isoindoline-1,3-dione (1 g,2.211 mmol), 6-oxa-3-azabicyclo[3.1.1]heptane HCl (180 mg, 1.328 mmol),cesium carbonate (2.161 g, 6.633 mmol), Pd2(dba)3 (202.5 mg, 0.2211mmol) and rac-BINAP (275.3 mg, 0.4422 mmol) in dioxane (5 mL) wasstirred overnight at 70° C., then heated in a microwave reactor for 15min at 150° C. The reaction was then diluted with methylene chloride,filtered though Celite, and concentrated. Silica gel flash columnchromatography (0-5% MeOH/DCM) yielded2-[4-[7-(6-oxa-3-azabicyclo[3.1.1]heptan-3-yl)quinoxalin-5-yl]oxycyclohexyl]isoindoline-1,3-dione(750 mg, 72.1%) as a yellow solid that was carried on to the nextreaction.

To a solution of2-[4-[7-(6-oxa-3-azabicyclo[3.1.1]heptan-3-yl)quinoxalin-5-yl]oxycyclohexyl]isoindoline-1,3-dione(800 mg, 1.700 mmol) in EtOH (10 mL) was added hydrazine monohydrate(85.10 mg, 83.35 μL, 1.700 mmol) and the reaction was stirred at refluxovernight, then concentrated, diluted with DCM, and filtered. Thefiltrate was concentrated, and purified on a 40 g silica gel cartridgewith 0-50% (20% NH3/MeOH) to yield4-[7-(6-oxa-3-azabicyclo[3.1.1]heptan-3-yl)quinoxalin-5-yl]oxycyclohexanamine(450 mg, 77.8%) as a yellow solid. 1H NMR (300 MHz, Chloroform-d) δ 8.65(d, J=1.9 Hz, 1H), 8.53 (d, J=1.9 Hz, 1H), 6.83 (q, J=2.6 Hz, 2H), 4.83(t, J=6.0 Hz, 4H), 3.87-3.60 (m, 5H), 3.34 (dt, J=8.7, 6.6 Hz, 1H),3.01-2.83 (m, 1H), 2.23 (dq, J=11.3, 5.8, 4.8 Hz, 2H), 2.07 (d, J=8.7Hz, 1H), 1.92-1.62 (m, 6H).

A mixture of4-[7-(6-oxa-3-azabicyclo[3.1.1]heptan-3-yl)quinoxalin-5-yl]oxycyclohexanamine(190 mg, 0.5581 mmol), 2-fluoropyrimidine (60 mg, 0.6118 mmol) and DIEA(200 μL, 1.148 mmol) in 2-propanol (2 mL) was heated in a microwavereactor for 20 min at 150° C. The reaction mixture was concentrated, andthen purified from 12 g silica gel cartridge with 0-6% MeOH/DCM to yieldN-[4-[7-(6-oxa-3-azabicyclo[3.1.1]heptan-3-yl)quinoxalin-5-yl]oxycyclohexyl]pyrimidin-2-amine(120.2 mg, 48.9%) as a yellow solid. Mass+1: 419.23; Retention Time:0.72; NMR Annotation: 1H NMR (400 MHz, Chloroform-d) δ 8.42 (d, J=1.9Hz, 1H), 8.30 (d, J=1.9 Hz, 1H), 8.04 (d, J=4.8 Hz, 2H), 6.65-6.56 (m,2H), 6.28 (t, J=4.8 Hz, 1H), 4.99 (d, J=8.1 Hz, 1H), 4.60 (d, J=6.5 Hz,3H), 3.79 (dd, J=8.2, 4.0 Hz, 0H), 3.62-3.38 (m, 4H), 3.17-3.03 (m, 1H),2.07-1.90 (m, 2H), 1.89-1.59 (m, 7H).

EXAMPLE 11 Preparation of4-methyl-N-[4-[(6-morpholino-2,1,3-benzoxadiazol-4-yl)oxy]cyclohexyl]pyrimidin-2-amine(Compound 665)

A mixture of 4,6-dibromo-2,1,3-benzoxadiazole (3 g, 10.80 mmol) andtert-butyl N-(4-hydroxycyclohexyl)carbamate (2.5 g, 11.61 mmol) wasdissolved in THF (60 mL) and cooled down to −30° C. To the mixture wasdropwise added sodium bis(trimethylsilyl)amide (NaHMDS, 23 mL of 1 M,23.00 mmol) over 10 min and the solution became dark blue. The mixturewas stirred for 30 min −20 to 0° C. and 30 min at rt. LC-MS showeddesired product peak only with no starting material. Cooled down to 0°C., quenched with 10% citric acid solution, extracted with EtOAc, driedover Na₂SO₄, and concentrated. Purified by 0-40% EtOAc/heptane.Recovered 1.9 g (42%) of tert-butyl((1s,4s)-4-((6-bromobenzo[c][1,2,5]oxadiazol-4-yl)oxy)cyclohexyl)carbamate(C17H22BrN3O4).

A mixture of tert-butylN-[4-[(6-morpholino-2,1,3-benzoxadiazol-4-yl)oxy]cyclohexyl]carbamate(2.6 g, 6.306 mmol), cesium carbonate (4 g, 12.28 mmol) ,diacetoxypalladium (140 mg, 0.6236 mmol), RuPhos (580 mg, 1.243 mmol)and morpholine (900 μL, 10.32 mmol) in dioxane (30 mL) purged with N2for 5 minutes. The mixture was stirred for 3 h at 90° C. LC-MS showeddesired product. The mixture was diluted with DCM, filtered though alayer of celite, concentrated and purified with a 80 g silica gelcartridge eluting with 0-80% EtOAc/heptane, recovering tert-butylN-[4-[(6-morpholino-2,1,3-benzoxadiazol-4-yl)oxy]cyclohexyl]carbamate(2.1 g, 80%). 1H NMR (400 MHz, Chloroform-d) δ 6.42 (dd, J=12.3, 1.7 Hz,2H), 4.86 (s, 1H), 4.58 (s, 1H), 4.00-3.86 (m, 4H), 3.38-3.18 (m, 4H),2.28-2.08 (m, 2H), 1.94-1.62 (m, 6H), 1.42 (s, 9H).

tert-butylN-[4-[(6-morpholino-2,1,3-benzoxadiazol-4-yl)oxy]cyclohexyl]carbamate

A solution of tert-butylN-[4-[(6-morpholino-2,1,3-benzoxadiazol-4-yl)oxy]cyclohexyl]carbamate(3.5 g, 8.363 mmol) i n DCM (30 mL) was added TFA (approximately 953.6mg, 644.3 μL, 8.363 mmol) and stirred for 1 hr. The mixture wasconcentrated and purified with 40 g silica gel cartridge eluting with0-10% (7N NH3/MeOH/DCM). Recovered4-[(6-morpholino-2,1,3-benzoxadiazol-4-yl)oxy]cyclohexanamine (2.5 g,94%) ESI-MS m/z calc. 318.1692, found 319.2 (M+1)⁺; Retention time: 0.55minutes.

A microwave vial was charged with4-[(6-morpholino-2,1,3-benzoxadiazol-4-yl)oxy]cyclohexanamine (1 g,3.141 mmol), 2-chloro-4-methyl-pyrimidine (600 mg, 4.667 mmol), RockPhosG3 (80 mg, 0.3175 mmol) and dioxane (10 mL). NaOtBu (4 mL of 2 M, 8.000mmol) was added to the mixture. The mixture was stirred for 15 minutesat 70° C. LC-MS showed a conversion, affording4-methyl-N-[4-[(6-morpholino-2,1,3-benzoxadiazol-4-yl)oxy]cyclohexyl]pyrimidin-2-amineThe mixture was diluted with DCM, filtered though a layer of celite,concentrated, and purified with 40 g silica gel cartridge eluting with0-10% MeOH/DCM. recovered the product. The product was triturated withEt₂O and dried under vacuum.4-methyl-N-[4-[(6-morpholino-2,1,3-benzoxadiazol-4-yl)oxy]cyclohexyl]pyrimidin-2-amine(795.3 mg, 59%), 1H NMR (400 MHz, Chloroform-d) δ 8.16 (d, J=5.1 Hz,1H), 6.49 (d, J=1.7 Hz, 1H), 6.47-6.40 (m, 2H), 5.07 (d, J=8.0 Hz, 1H),4.95 (s, 1H), 3.96-3.85 (m, 4H), 3.32-3.24 (m, 4H), 2.34 (s, 3H),2.25-2.08 (m, 2H), 2.04-1.74 (m, 6H). ESI-MS m/z calc. 410.20663, found411.3 (M+1)⁺; Retention time: 0.63 minutes.

Preparation ofN-methyl-2-((4-((6-morpholinobenzo[c][1,2,5]oxadiazol-4-yl)oxy)cyclohexyl)amino)pyrimidine-4-carboxamide(Compound (666))

To a mixture of 4,6-dibromo-2,1,3-benzoxadiazole (1 g, 3.598 mmol),(4-methoxyphenyl)methanol (500 μL, 4.010 mmol) in THF (20 mL) at −78° C.was dropwise added NaHMDS (4 mL of 1 M, 4.000 mmol). The solution becameblue. The mixture was stirred at −78° C. for 30 minutes. The mixture wasallowed to warm up to RT and was atirred for 1 h at RT. LC-MS showed aproduct peak and no starting material. TLC showed a spot slightly lesspolar than the starting material. The reaction was quenched with sat.NH₄Cl, extracted with EtOAc, washed with H₂O, and dried over Na₂SO₄,concentrated. The product was purified with 40 g silica gel cartridgeeluting with 0-30% EtOAc, recovering6-bromo-4-[(4-methoxyphenyl)methoxy]-2,1,3-benzoxadiazole (850 mg, 70%)as slightly yellow solid. 1H NMR (300 MHz, CDCl3) δ 7.48 (d, J=1.2 Hz,1H), 7.38-7.19 (m, 2H), 6.94-6.74 (m, 2H), 6.57 (d, J=1.1 Hz, 1H), 5.11(s, 2H), 3.68 (d, J=9.4 Hz, 3H).

A mixture of 6-bromo-4-[(4-methoxyphenyl)methoxy]-2,1,3-benzoxadiazole(830 mg, 2.476 mmol), cesium carbonate (2.4 g, 7.366 mmol), Pd(OAc)2 (50mg, 0.2227 mmol), rac-BINAP (300 mg, 0.4818 mmol) and morpholine (300μL, 3.440 mmol) in dioxane (10 mL) was bubbled with N₂ for 10 minutes.The mixture was heated to 80° C. The mixture was stirred for 4.5 h at80° C. LC-MS showed a clean product. The mixture was cooled to rt,diluted with DCM, filtered though a layer of celite, and concentrated.The product was purified with 4 0 g silica gel cartridge with 0-70%EtOAc/DCM, recovered4-[(4-methoxyphenyl)methoxy]-6-morpholino-2,1,3-benzoxadiazole (750 mg,89%) . 1H NMR (300 MHz, CDCl3) δ 7.42 (d, J=8.7 Hz, 2H), 7.03-6.87 (m,2H), 6.48 (d, J=1.6 Hz, 1H), 6.40 (d, J=1.6 Hz, 1H), 5.30 (d, J=13.5 Hz,2H), 3.99-3.75 (m, 7H), 3.31-3.17 (m, 4H).

A solution of4-[(4-methoxyphenyl)methoxy]-6-morpholino-2,1,3-benzoxadiazole (750 mg,2.197 mmol) in DCM (10 mL) was added TFA (2 mL, 25.96 mmol) and stirredfor 1 h. The mixture was concentrated and the product purified with 12 gsilica gel cartridge eluting with 0-10% MeOH/DCM.6-Morpholino-2,1,3-benzoxadiazol-4-ol (400 mg, 82%) is recovered as ayellow foam.

Amixture of 6-morpholino-2,1,3-benzoxadiazol-4-ol (50 mg, 0.2260 mmol) ,[4-[[4-(methylcarbamoyl)pyrimidin-2-yl]amino]DMF, hexyl]methanesulfonate (240 mg, 0.7308 mmol) , and cesium carbonate (250 mg,0.7673 mmol) in DMF (1 mL) was stirred overnight at 80° C. LC-MS showedthe desired product. The mixture was diluted with DCM and H₂O, extractedwith DCM, dried over Na₂SO₄, concentrated. The product was purified witha 4 g silica gel cartridge eluting with 0-5% MeOH/DCM, recovering thedesired productN-methyl-2-((4-((6-morpholinobenzo[c][1,2,5]oxadiazol-4-yl)oxy)cyclohexyl)amino)pyrimidine-4-carboxamide(41.8 mg, 39%) . 1H NMR (300 MHz, CDCl₃) δ 8.50 (d, J=4.9 Hz, 1H), 7.76(s, 1H), 7.34 (d, J=4.9 Hz, 1H), 6.50 (d, J=1.3 Hz, 1H), 6.43 (d, J=1.5Hz, 1H), 5.21 (d, J=7.2 Hz, 1H), 4.96 (s, 1H), 4.04 (s, 1H), 3.94-3.81(m, 4H), 3.33-3.17 (m, 4H), 3.01 (t, J=9.9 Hz, 3H), 2.19 (d, J=8.2 Hz,2H), 2.06-1.78 (m, 6H). ESI-MS m/z calc. 453.21246, found 454.39 (M+1)⁺;Retention time: 0.72 minutes.

Tables 1 and 2 provides analytical characterization data for certaincompounds of formula (III-E-1 and III-E-2) (blank cells indicate thatthe test was not performed).

Table 1 ¹ H NMR (300 MHz, unless Cmpd. ESMS indicated otherwise) No.Compound Structure (M + H) NMR peaks given as δ values 18

406.48 (CDCl₃) δ 8.65 (d, J = 2.0 Hz, 1H), 8.37 (d, J = 2.0 Hz, 1H),8.27 (d, J = 4.8 Hz, 2H), 6.60 (d, J = 2.4 Hz, 1H), 6.51 (t, J = 4.8 Hz,1H), 6.36 (d, J = 2.4 Hz, 1H), 6.15 (d, J = 7.8 Hz, 1H), 5.20 (d, J =7.7 Hz, 1H), 4.04 (d, J = 7.9 Hz, 1H), 3.96-3.82 (m, 4H), 3.70 (s, 1H),3.39-3.24 (m, 4H), 1.94 (dd, J = 13.7, 4.4 Hz, 6H), 1.78 (dt, J = 28.8,16.1 Hz, 2H) 23

396.2 (CDCl₃) δ 8.20 (d, J = 4.9 Hz, 2H), 6.46 (t, J = 4.8 Hz, 1H), 6.05(d, J = 1.6 Hz, 1H), 5.82 (s, 1H), 5.24 (s, 1H), 4.82 (d, J = 7.0 Hz,1H), 3.98 (s, 1H), 3.85-3.72 (m, 4H), 3.60 (s, 1H), 3.23-3.06 (m, 4H),1.95- 1.62 (m, 8H). [2] 24

405.59 (400 MHz, CDCl₃) δ 8.65 (s, 1H), 8.37 (d, J = 1.8 Hz, 1H), 8.08(d, J = 4.9 Hz, 1H), 7.41 (t, J = 7.7 Hz, 1H), 6.61 (s, 1H), 6.58-6.53(m, 1H), 6.46-6.30(m, 2H), 6.15 (d, J = 7.3 Hz, 1H), 4.56 (s, 1H),3.98-3.78 (m, 5H), 3.76-3.61 (m, 1H), 3.42-3.24 (m, 4H), 1.97 (d, J =29.6 Hz, 6H), 1.86-1.66 (m, 2H) 27

406.58 (CDCl₃) δ 8.65 (d, J = 2.0 Hz, 1H), 8.36 (d, J = 2.0 Hz, 1H),7.98 (dd, J = 2.8, 1.5 Hz, 1H), 7.88 (d, J = 1.5 Hz, 1H), 7.79 (d, J =2.8 Hz, 1H), 6.62 (d, J = 2.4 Hz, 1H), 6.37 (d, J = 2.4 Hz, 1H), 6.17(s, 1H), 8.69-8.61 (m, 1H), 4.60 (d, J = 7.7 Hz, 1H), 4.08-3.83 (m, 5H),8.71-8.57 (m, 1H), 3.73 (t, J = 6.9 Hz, 1H), 3.40-3.25 (m, 4H), 1.96 (h,J = 4.9 Hz, 6H), 1.77 (q, J = 7.4, 6.1 Hz, 2H) 28

407.3 (400 MHz, CDCl₃) δ 8.77- 8.59 (m, 2H), 8.29 (d, J = 4.9 Hz, 2H),7.01-6.87 (m, 2H), 6.61-6.48 (m, 1H), 4.82 (s, 1H), 4.05 (s, 1H), 3.93(t, J = 4.8 Hz, 4H), 3.35 (t, J = 4.8 Hz, 4H), 2.23 (d, J = 13.1 Hz,2H), 2.05-1.82 (m, 6H) 29

450.61 (DMSO-d₆) δ 12.65 (s, 1H), 8.69 (d, J = 2.0 Hz, 2H), 8.43 (d, J =2.0 Hz, 1H), 8.00 (d, J = 7.9 Hz, 1H), 6.54 (d, J = 2.5 Hz, 1H), 6.48(d, J = 2.3 Hz, 1H), 6.17 (d, J = 8.2 Hz, 1H), 4.06 (s, 1H), 3.77 (dd, J= 5.9, 3.8 Hz, 4H), 3.29 (s, 5H), 2.04-1.46 (m, 8H) 30

406.52 (CDCl₃) δ 8.66 (d, J = 2.0 Hz, 1H), 8.58 (s, 1H), 8.37 (d, J =2.0 Hz, 1H), 8.13 (s, 2H), 6.63 (d, J = 2.4 Hz, 1H), 6.37 (d, J = 2.4Hz, 1H), 6.14 (d, J = 7.6 Hz, 1H), 3.96-3.86 (m, 4H), 3.76 (d, J = 7.7Hz, 2H), 3.54 (d, J = 8.3 Hz, 1H), 3.39-3.29 (m, 4H), 2.02-1.86 (m, 6H),1.76 (q, J = 8.9, 8.3 Hz, 2H) 32

464.6 (CDCl₃) δ 8.78 (d, J = 1.3 Hz, 1H), 8.66 (d, J = 2.0 Hz, 1H), 8.37(d, J = 2.0 Hz, 1H), 7.91 (d, J = 1.3 Hz, 1H), 6.62 (d, J = 2.4 Hz, 1H),6.37 (d, J = 2.4 Hz, 1H), 6.16 (d, J = 7.6 Hz, 1H), 5.13 (d, J = 7.6 Hz,1H), 4.10 (s, 1H), 3.99-3.86 (m, 7H), 3.76 (s, 1H), 3.39- 3.28 (m, 4H),1.98 (h, J = 4.8 Hz, 6H), 1.80 (t, J = 8.7 Hz, 2H) 33

407.3 (CDCl₃) δ 8.63 (d, J = 1.9 Hz, 1H), 7.99 (dd, J = 2.8, 1.5 Hz,1H), 7.94-7.85 (m, 1H), 7.79 (d, J = 2.8 Hz, 1H), 7.14 (d, J = 1.0 Hz,1H), 7.03-6.87 (m, 2H), 4.82 (d, J = 5.7 Hz, 1H), 4.70 (d, J = 8.0 Hz,1H), 4.03-3.86 (m, 4H), 3.51 (s, 1H), 3.43-3.30 (m, 4H), 2.35-1.81 (m,8H) 34

408.5 (CDCl₃) δ 8.69 (dd, J = 3.4, 1.9 Hz, 1H), 8.62 (dd, J = 3.6, 1.9Hz, 1H), 8.51 (dd, J = 4.8, 2.2 Hz, 2H), 7.01- 6.83 (m, 3H), 5.18 (tt, J= 7.0, 3.4 Hz, 1H), 4.79 (tt, J = 6.9, 3.1 Hz, 1H), 4.00-3.85 (m, 4H),3.34 (dq, J = 4.8, 2.6 Hz, 4H), 2.44-2.16 (m, 4H), 1.92 (tdd, J = 16.4,7.7, 2.8 Hz, 4H) 36

404.2 (CDCl₃) δ 8.94-8.76 (m, 2H), 8.29 (d, J = 4.8 Hz, 2H), 7.67 (d, J= 1.7 Hz, 1H), 6.53 (t, J = 4.8 Hz, 1H), 6.37 (tt, J = 3.1, 1.5 Hz, 1H),5.30 (d, J = 7.9 Hz, 1H), 4.87 (dt, J = 7.5, 3.6 Hz, 1H), 4.43 (q, J =2.8 Hz, 2H), 4.02 (t, J = 5.5 Hz, 3H), 2.68 (dqd, J = 6.0, 3.4, 3.0, 1.8Hz, 2H), 2.35- 2.11 (m, 2H), 2.07-1.84 (m, 6H) 37

425.25 (CDCl₃) δ 8.71 (d, J = 1.9 Hz, 1H), 8.63 (d, J = 1.9 Hz, 1H),8.18 (dd, J = 3.7, 0.8 Hz, 2H), 7.01-6.85 (m, 2H), 5.37-5.20 (m, 1H),4.79 (d, J = 5.5 Hz, 1H), 4.02-3.85 (m, 4H), 3.43- 3.29 (m, 4H),2.31-2.15 (m, 2H), 2.02-1.85 (m, 6H) 38

407.25 (CDCl₃) δ 8.61 (d, J = 2.0 Hz, 1H), 8.55 (d, J = 1.9 Hz, 1H),8.20 (d, J = 4.8 Hz, 2H), 6.87 (d, J = 2.5 Hz, 1H), 6.81 (d, J = 2.5 Hz,1H), 6.46 (t, J = 4.8 Hz, 1H), 4.95 (s, 1H), 4.45 (tt, J = 10.7, 3.6 Hz,1H), 3.95-3.77 (m, 5H), 3.32- 3.19 (m, 4H), 2.34-2.10 (m, 4H), 1.82 (dt,J = 12.9, 10.0 Hz, 2H), 1.45-1.20 (m, 2H) 39

441.28 (CDCl₃) δ 8.72 (d, J = 1.9 Hz, 1H), 8.62 (d, J = 1.9 Hz, 1H),8.37 (s, 1H), 6.98 (d, J = 2.5 1H), 6.36 (d, J = 1.0 Hz, 1H), 4.91-4.76(m, 1H), 4.00-3.88 (m, 4H), 3.45-3.24 (m, 4H), 2.34-2.17 (m, 2H),2.03-1.84 (m, 6H) 40

441.3 (CDCl₃) δ 8.71 (d, J = 1.9 Hz, 1H), 8.61 (d, J = 1.9 Hz, 1H), 7.15(d, J = 9.3 Hz, 1H), 7.00- 6.87 (m, 2H), 6.64 (d, J = 9.3 Hz, 1H),4.89-4.76 (m, 2H), 4.11-4.03 (m, 1H), 4.00-3.83 (m, 4H), 3.40-3.24 (m,4H), 2.23 (dq, J = 12.9, 6.3, 5.6 Hz, 2H), 2.02-1.79 (m, 6H) 41

421.43 (CDCl₃) δ 8.63 (d, J = 1.9 Hz, 1H), 8.55 (d, J = 1.9 Hz, 1H),7.58 (d, J = 23.6 Hz, 2H), 6.89 (d, J = 2.4 Hz, 1H), 6.84 (d, J = 2.5Hz, 1H), 4.73 (s, 2H), 3.93-3.72 (m, 5H), 3.34- 3.18 (m, 4H), 2.29 (s,3H), 2.15 (m, 2H), 1.84 (m, 6H) 42

408.56 (CDCl₃) δ 8.70 (d, J = 1.9 Hz, 1H), 8.63 (d, J = 1.9 Hz, 1H),8.52 (d, J = 4.8 Hz, 2H), 7.01-6.87 (m, 3H), 5.17 (ddt, J = 8.7, 6.7,3.4 Hz, 1H), 4.76-4.58 (m, 1H), 4.00-3.87 (m, 4H), 3.40-3.27 (m, 4H),2.43-2.22 (m, 4H), 2.05-1.87 (m, 2H), 1.86-1.71 (m, 2H) 44

432.6 (CDCl₃) δ 8.71 (d, J = 2.0 Hz, 1H), 8.64 (d, J = 2.0 Hz, 1H), 8.54(d, J = 2.9 Hz, 1H), 8.47 (d, J = 3.0 Hz, 1H), 6.99 (d, J = 2.4 Hz, 1H),6.92 (d, J = 2.5 Hz, 1H), 5.81 (d, J = 8.3 Hz, 1H), 4.84 (dt, J = 5.3,2.8 Hz, 1H), 4.13-4.05 (m, 1H), 4.00-3.84 (m, 4H), 3.43-3.30 (m, 4H),2.32-2.17 (m, 2H), 2.02-1.85 (m, 6H) 45

485.26 (CDCl₃) δ 8.70 (d, J = 2.0 Hz, 1H), 8.64 (d, J = 1.9 Hz, 1H),8.29 (s, 2H), 6.98 (d, J = 2.5 Hz, 1H), 6.92 (d, J = 2.5 Hz, 1H), 5.29(d, J = 8.3 Hz, 1H), 4.81 (s, 1H), 4.04-3.84 (m, 4H), 3.42-3.31 (m, 4H),2.22 (s, 2H), 1.92 (d, J = 4.9 Hz, 6H) 46

407.57 (CDCl₃) δ 8.63 (d, J = 2.0 Hz, 1H), 8.52 (d, J = 4.8 Hz, 2H),8.37 (d, J = 2.0 Hz, 1H), 6.92 (t, J = 4.8 Hz, 1H), 6.62 (d, J = 2.4 Hz,1H), 6.38 (d, J = 2.4 Hz, 1H), 6.16 (s, 1H), 5.27 (s, 1H), 4.06-3.78 (m,4H), 3.64 (s, 1H), 3.48-3.20 (m, 4H), 2.14 (s, 2H), 2.04- 1.80 (m, 4H)49

425.39 (400 MHz, CDCl₃) δ 8.70 (d, J = 1.9 Hz, 1H), 8.60 (d, J = 1.9 Hz,1H), 7.99 (s, 1H), 6.96 (d, J = 2.4 Hz, 1H), 6.89 (d, J = 2.3 Hz, 1H),6.20 (s, 1H), 5.19 (bs, 1H), 4.81 (bs, 1H), 3.96-3.84 (m, 4H), 3.40-3.27(m, 4H), 2.29-2.14 (m, 2H), 1.99-1.81 (m, 6H) 50

425.39 (400 MHz, CDCl₃) δ 8.69 (d, J = 1.9 Hz, 1H), 8.60 (d, J = 1.9 Hz,1H), 8.30 (d, J = 2.1 Hz, 1H), 6.95 (d, J = 2.4 Hz, 1H), 6.89 (d, J =2.4 Hz, 1H), 5.84 (s, 1H), 5.42 (s, 1H), 4.81 (s, 1H), 3.99-3.82 (m,4H), 3.39-3.24 (m, 4H), 2.31-2.19 (m, 2H), 2.08-1.72 (m, 8H) 51

425.33 (400 MHz, CDCl₃) δ 8.69 (d, J = 1.9 Hz, 1H), 8.60 (d, J = 1.9 Hz,1H), 8.30 (d, J = 2.1 Hz, 1H), 6.95 (d, J = 2.4 Hz, 1H), 6.89 (d, J =2.4 Hz, 1H), 5.84 (s, 1H), 5.42 (s, 1H), 4.81 (s, 1H), 3.99-3.82 (m,4H), 3.39-3.24 (m, 4H), 2.31- 2.19 (m, 2H), 2.08-1.72 (m, 8H) 52

435.19 (400 MHz, CDCl₃) δ 8.68 (d, J = 1.4 Hz, 1H), 8.60 (d, J = 1.5 Hz,1H), 6.93 (s, 1H), 6.90 (s, 1H), 6.28 (s, 1H), 5.06 (d, J = 7.9 Hz, 1H),4.77 (s, 1H), 4.06 (bs, 1H), 3.97- 3.84 (m, 4H), 3.38-3.25 (m, 4H), 2.27(s, 6H), 2.18-2.09 (m, 2H), 1.94-1.83 (m, 7H) 53

433.25 (CDCl₃) δ 9 Hz, 1H), 8.28 (d, J = 4.8 Hz, 2H), 6.85 (t, J = 1.9Hz, 2H), 6.51 (t, J = 4.8 Hz, 1H), 5.29 (d, J = 6.3 Hz, 1H), 4.80 (dq, J= 5.5, 2.8 Hz, 1H), 4.57 (d, J = 3.9 Hz, 2H), 4.02 (t, J = 6.2 Hz, 1H),3.54-3.43 (m, 2H), 3.19 (dd, J = 11.6, 2.6 Hz, 2H), 2.27- 2.14 (m, 2H),2.08-1.85 (m, 10H) 54

437.27 (CDCl₃) 6 8.70 (d, J = 1.9 Hz, 1H), 8.62 (d, J = 1.9 Hz, 1H),7.45 (d, J = 7.8 Hz, 2H), 7.02- 6.81 (m, 2H), 4.78 (ddd, J = 7.3, 5.6,3.1 Hz, 1H), 4.66 (d, J = 7.9 Hz, 1H), 4.01-3.78 (m, 7H), 3.41-3.25 (m,4H), 2.30- 2.08 (m, 2H), 1.94 (h, J = 8.8, 8.2 Hz, 6H) 55

432.3 (CDCl₃) 6 8.73 (d, J = 2.0 Hz, 1H), 8.63 (d, J = 2.0 Hz, 1H), 7.42(d, J = 9.3 Hz, 1H), 6.96 (dd, J = 19.6, 2.5 Hz, 2H), 6.65 (d, J = 9.3Hz, 1H), 5.40 (s, 1H), 4.86 (s, 1H), 3.94 (dd, J = 5.9, 3.8 Hz, 4H),3.37 (dd, J = 6.0, 3.7 Hz, 4H), 2.27 (d, J = 12.7 Hz, 2H), 2.05-1.80(m,6H) 59

431.19 (400 MHz, CDCl₃) δ 8.70 (d, J = 1.9 Hz, 1H), 8.61 (d, J = 1.9 Hz,1H), 8.36 (d, J = 1.8 Hz, 1H), 7.54 (dd, J = 8.8, 2.2 Hz, 1H), 6.96 (d,J = 2.4 Hz, 1H), 6.90 (d, J = 2.4 Hz, 1H), 6.37 (d, J = 8.7 Hz, 1H),5.11 (s, 1H), 4.80 (s, 1H), 3.94 (s, 1H), 3.94-3.79 (m, 4H), 3.38-3.25(m, 4H), 2.32- 2.12 (m, 2H), 2.02-1.78 (m, 6H) 60

425.23 (CDCl₃) δ 8.71 (d, J = 1.9 Hz, 1H), 8.62 (d, J = 1.9 Hz, 1H),7.06-6.88 (m, 3H), 6.80 (dd, J = 9.4, 6.3 Hz, 1H), 4.97- 4.71 (m, 2H),4.14 (q, J = 7.1 Hz, 1H), 4.01-3.85 (m, 4H), 3.44-3.24 (m, 4H), 2.23 (d,J = 10.7 Hz, 2H), 1.96 (dt, J = 11.0, 7.6 Hz, 6H) 61

397.15 (400 MHz, CDCl₃) δ 8.69 (d, J = 1.8 Hz, 1H), 8.61 (d, J = 1.9 Hz,1H), 6.94 (d, J = 2.3 Hz, 1H), 6.87 (s, 1H), 5.78- 5.64 (m, 1H), 4.73(s, 1H), 4.01 (s, 1H), 3.97-3.78 (m, 4H), 3.43-3.18 (m, 4H), 2.26-2.05(m, 2H), 1.98-1.73 (m, 6H), 1.36-1.26 (m, 1H), 1.01-0.92 (m, 2H),0.78-0.67 (m, 2H) 63

463.54 (CDCl₃) δ 8.53 (s, 1H), 8.29 (d, J = 4.8 Hz, 2H), 6.92 (d, J =2.5 Hz, 1H), 6.85 (d, J = 2.5 Hz, 1H), 6.52 (t, J = 4.8 Hz, 1H), 5.25(d, J = 8.3 Hz, 1H), 4.79 (s, 1H), 4.07 (d, J = 20.4 Hz, 1H), 3.98-3.85(m, 4H), 3.43-3.21 (m, 4H), 3.05-2.83 (m, 2H), 2.30-2.14 (m, 1H),2.03-1.71 (m, 7H), 1.45 (dq, J = 14.5, 7.3 Hz, 2H), 0.98 (t, J = 7.3 Hz,3H) 64

541.26 (CDCl₃) δ 8.55 (s, 1H), 8.29 (d, J = 1.5 Hz, 2H), 7.05-6.93 (m,1H), 6.85 (d, J = 2.5 Hz, 1H), 5.49 (d, J = 8.0 Hz, 1H), 4.80 (d, J =5.8 Hz, 1H), 4.03-3.81 (m, 5H), 3.45-3.27 (m, 4H), 2.98 (dd, J = 8.5,7.0 Hz, 2H), 2.32-2.09 (m, 2H), 2.00-1.71 (m, 8H), 1.56-1.34 (m, 2H),0.98 (td, J = 7.3, 3.3 Hz, 3H) 65

450.49 (CDCl₃) δ 8.76-8.66 (m, 3H), 8.61 (d, J = 1.9 Hz, 1H), 6.99- 6.87(m, 2H), 5.71 (d, J = 8.1 Hz, 1H), 4.81 (s, 1H), 4.11 (s, 1H), 3.92 (t,J = 4.9 Hz, 4H), 3.34 (t, J = 4.9 Hz, 4H), 2.22 (d, J = 10.2 Hz, 2H),2.02- 1.85 (m, 6H) 66

495.23 (CDCl₃) δ 8.77 (d, J = 2.3 Hz, 1H), 8.72 (d, J = 2.3 Hz, 1H),7.12 (d, J = 2.4 Hz, 1H), 7.02 (d, J = 2.4 Hz, 1H), 4.92 (s, 1H),4.18-4.08 (m, 2H), 3.93 (t, J = 4.9 Hz, 4H), 3.85-3.76 2H), 3.47 (t, J =4.9 Hz, 4H), 2.26 (d, J = 13.3 Hz, 2H), 2.07 (t, J = 10.5 Hz, 2H),2.01-1.72 (m, 2H), 1.26 (t, J = 7.0 Hz, 67

450.3 (CDCl₃) δ 8.71 (d, J = 2.0 Hz, 1H), 8.64 (d, J = 1.9 Hz, 1H), 8.02(s, 1H), 6.94 (dd, J = 12.2, 2.5 Hz, 2H), 5.32 (s, 2H), 4.87 (d, J = 8.1Hz, 1H), 4.79 (s, 1H), 4.01-3.86 (m, 4H), 3.36 J = 5.4, 4.7 Hz, 4H),2.83 (s, 6H), 2.19 (s, 2H), 1.92 (d, J = 4.8 Hz, 6H) 69

451.21 (CDCl₃) δ 9.05 (s, 1H), 8.88- 8.71 (m, 3H), 8.49 (s, 1H), 7.06(d, J = 2.3 Hz, 1H), 6.96 (d, J = 2.3 Hz, 1H), 4.85 (s, 1H), 4.17 (s,1H), 3.93 (t, J = 4.8 Hz, 4H), 3.42 (t, J = 4.9 Hz, 4H), 2.25-2.10 (m,2H), 1.95 (d, J = 11.9 Hz, 4H) 71

464.4 (CDCl₃) δ 8.79 - 8.64 (m, 3H), 8.59 (d, J = 1.9 Hz, 1H), 6.99-6.88 (m, 2H), 6.19 (q, J = 4.7 Hz, 1H), 5.90 (d, J = 8.2 Hz, 1H), 4.81(dq, J = 5.3, 2.7 Hz, 1H), 4.08 (qd, J = 8.2, 6.5, 2.3 Hz, 1H),3.97-3.87 (m, 4H), 3.39-3.29 (m, 4H), 2.93 (d, J = 4.8 Hz, 3H),2.27-2.14 (m, 2H), 2.06-1.79 (m, 6H) 72

478.39 (CDCl₃) δ 3.97-3.87 (m, 4H), 3.39-3.29 (m, 4H), 3.10 (s, 6H),2.22 (dt, J = 11.3, 5.1 Hz, 2H), 1.94 (dd, J = 8.3, 3.9 Hz, 6H),4.12-4.01 (m, 1H), 8.70 (d, J = 1.9 Hz, 1H), 8.62 (d, J = 1.9 Hz, 1H),8.46 (s, 2H), 7.00-6.87 (m, 2H), 5.57 (d, J = 8.1 Hz, 1H), 4.81 (dq, J =5.1, 2.4 Hz, 1H) 84

431.19 (400 MHz, CDCl₃) δ 8.69 (d, J = 1.9 Hz, 1H), 8.61 (d, J = 1.9 Hz,1H), 8.19 (d, J = 4.8 Hz, 1H), 6.95 (d, J = 2.3 Hz, 1H), 6.90 (d, J =2.4 Hz, 1H), 6.70 (dd, J = 5.1, 1.2 Hz, 1H), 6.56 (s, 1H), 4.87 (d, J =7.6 Hz, 1H), 4.80 (s, 1H), 3.96- 3.88 (m, 4H), 3.85 (s, 1H), 3.38-3.28(m, 4H), 2.28-2.14 (m, 2H), 2.00-1.85 (m, 6H) 87

431.2 (CDCl₃) δ 8.63 (d, J = 1.9 Hz, 1H), 8.57 (d, J = 1.9 Hz, 1H), 8.20(dd, J = 5.0, 1.9 Hz, 1H), 7.57 (dd, J = 7.6, 1.9 Hz, 1H), 6.89 (d, J =2.5 Hz, 1H), 6.85 (d, J = 2.5 Hz, 1H), 6.51 (dd, J = 7.6, 4.9 Hz, 1H),5.12 (d, J = 7.7 Hz, 1H), 4.83-4.71 (m, 0H), 4.24-4.03 (m, 1H), 3.92-3.77 (m, 4H), 3.35-3.19 (m, 4H), 2.28-2.10 (m, 2H), 1.88 (td, J = 8.3,6.8, 3.9 Hz, 6H) 93

451.21 (methanol-d₄) δ 8.69 (d, J = 2.2 Hz, 1H), 8.59 (d, J = 2.2 Hz,1H), 8.53 (d, J = 4.9 Hz, 1H), 7.19 (d, J = 5.0 Hz, 1H), 7.15 (d, J =2.3 Hz, 1H), 6.87 (d, J = 2.4 Hz, 1H), 4.95 (d, J = 7.7 Hz, 1H), 4.10(s, 1H), 3.95-3.82 (m, 4H), 3.47-3.37 (m, 4H), 2.20 (d, J = 10.1 Hz,2H), 2.04-1.81 (m, 6H) 94

437.44 (CDCl₃) δ 8.68 (d, J = 2.0 Hz, 1H), 8.60 (d, J = 1.9 Hz, 1H),8.29 (s, 2H), 6.98-6.87 (m, 2H), 5.36 (d, J = 8.1 Hz, 1H), 4.79 (q, J =5.2, 4.0 Hz, 1H), 4.52 (s, 2H), 4.01 (dd, J = 8.1, 4.3 Hz, 1H),3.95-3.84 (m, 4H), 3.39-3.28 (m, 4H), 2.25- 2.12 (m, 2H), 1.99-1.82 (m,6H) 104

443.38 (CDCl₃) δ 8.73 (s, 1H), 8.68 (d, J = 2.4 Hz, 1H), 6.99 (d, J =2.4 Hz, 1H), 6.85 (d, J = 2.4 Hz, 1H), 5.69 (d, J = 2.5 Hz, 1H), 4.76(s, 1H), 3.84 (dd, J = 5.9, 3.9 Hz, 4H), 3.38 (dd, J = 6.0, 3.9 Hz, 4H),2.15 (d, J = 11.1 Hz, 2H), 1.96-1.67 (m, 6H) 108

478.26 (CDCl₃) δ 8.69 (d, J = 1.9 Hz, 1H), 8.61 (d, J = 2.0 Hz, 1H),8.49 (d, J = 1.4 Hz, 1H), 7.77 (d, J = 1.4 Hz, 1H), 6.93 (dd, J = 16.1,2.5 Hz, 2H), 5.03 (d, J = 7.9 Hz, 1H), 4.81 (td, J = 5.3, 2.6 Hz, 1H),4.09-3.98 (m, 1H), 3.98-3.87 (m, 4H), 3.39-3.26 (m, 4H), 3.19 (s, 3H),3.12 (s, 3H), 2.22 (dt, J = 11.2, 4.9 Hz, 2H), 2.02- 1.82 (m, 6H) 109

447.02 (CDCl₃) δ 8.70 (d, J = 1.9 Hz, 1H), 8.63 (d, J = 1.9 Hz, 1H),8.10 (d, J = 0.5 Hz, 2H), 6.97 (d, J = 2.5 Hz, 1H), 6.92 (d, J = 2.5 Hz,1H), 5.13 (d, J = 8.3 Hz, 1H), 4.80 (s, 1H), 4.06-3.86 (m, 5H), 3.35(dd, J = 5.9, 3.8 Hz, 4H), 2.27- 2.14 (m, 2H), 1.92 (d, J = 5.1 Hz, 6H),1.79-1.44 (m, 6H), 1.28 (t, J = 7.1 Hz, 1H), 1.03- 0.83 (m, 2H),0.68-0.51 (m, 2H) 134

437.3 (CDCl₃) δ 8.70 (d, J = 1.9 Hz, 1H), 8.63 (d, J = 1.9 Hz, 1H), 8.07(s, 2H), 6.97 (d, J = 2.5 Hz, 1H), 6.92 (d, J = 2.6 Hz, 1H), 4.99 (d, J= 8.0 Hz, 1H), 4.81 (d, J = 5.9 Hz, 1H), 3.93 (dd, J = 6.0, 3.7 Hz, 5H),3.81 (s, 3H), 3.45-3.24 (m, 4H), 2.21 (d, J = 8.6 Hz, 2H), 2.05- 1.78(m, 6H) 135

479.2 (CDCl₃) δ 8.70 (d, J = 1.9 Hz, 1H), 8.63 (d, J = 1.9 Hz, 1H), 8.19(s, 2H), 6.96 (d, J = 2.5 Hz, 1H), 6.92 (d, J = 2.5 Hz, 1H), 5.12 (d, J= 8.1 Hz, 1H), 4.81 (d, J = 5.6 Hz, 1H), 4.09- 3.85 (m, 5H), 3.63-3.41(m, 4H), 3.43-3.26 (m, 4H), 2.69 (t, J = 6.6 Hz, 2H), 2.33-2.13 (m, 2H),2.03-1.83 (m, 6H), 1.21 (t, J = 7.0 Hz, 3H) 139

450.17 (400 MHz, CDCl₃) δ 8.74 (d, J = 1.9 Hz, 2H), 8.70 (d, J = 1.9 Hz,1H), 8.03 (dd, J = 8.9, 2.0 Hz, 1H), 6.97 (d, J = 2.4 Hz, 1H), 6.92 (d,J = 2.4 Hz, 1H), 6.37 (d, J = 8.9 Hz, 1H), 6.05 (s, 1H), 4.81 (s, 1H),3.99-3.88 (m, 4H), 3.84 (s, 1H), 3.39-3.27 (m, 4H), 2.31-2.17 (m, 2H),2.08-1.98 (m, 2H), 1.98-1.82 (m, 4H) 140

449.19 (400 MHz, CDCl₃) δ 8.70 (d, J = 1.9 Hz, 1H), 8.61 (d, J = 1.9 Hz,1H), 8.53 (d, J = 2.1 Hz, 1H), 7.88 (dd, J = 8.8, 2.4 Hz, 1H), 6.95 (d,J = 2.4 Hz, 1H), 6.90 (d, J = 2.4 Hz, 1H), 6.39 (d, J = 8.7 Hz, 1H),5.61 (s, 2H), 4.97 (d, J = 7.9 Hz, 1H), 4.80 (s, 1H), 4.02- 3.82 (m,5H), 3.42-3.26 (m, 4H), 2.27-2.14 (m, 2H), 2.00-1.81 (m, 6H) 142

421.2 (CDCl₃) δ 8.70 (d, J = 1.9 Hz, 1H), 8.63 (d, J = 1.9 Hz, 1H), 8.14(d, J = 0.8 Hz, 2H), 7.01- 6.90 (m, 2H), 4.81 (td, J = 5.6, 2.7 Hz, 1H),4.08-3.84 (m, 5H), 3.43-3.26 (m, 4H), 2.25- 2.10 (m, 5H), 2.02-1.83 (m,6H) 143

422.25 (CDCl₃) δ 8.70 (d, J = 1.9 Hz, 1H), 8.63 (d, J = 1.9 Hz, 1H),8.02 (s, 2H), 6.95 (dd, J = 11.8, 2.5 Hz, 2H), 4.81 (s, 2H), 4.08-3.85(m, 5H), 3.41- 3.30 (m, 4H), 2.20 (d, J = 10.1 Hz, 2H), 1.95 (d, J =19.7 Hz, 6H), 1H NMR (300 MHz, Methanol-d4) 8.68 (d, J = 2.0 Hz, 1H),8.56 (d, J = 2.1 Hz, 1H), 7.93 (s, 2H), 7.11 (d, J = 2.5 Hz, 1H), 6.88(d, J = 2.4 Hz, 1H), 3.96-3.71 (m, 5H), 3.37 (dd, J = 5.8, 3.9 Hz, 4H),2.27-2.04 (m, 2H), 1.98-1.74 (m, 6H). [2] 144

463.2 (400 MHz, CDCl₃) δ 8.69 (d, J = 1.9 Hz, 1H), 8.61 (d, J = 1.9 Hz,1H), 8.47 (d, J = 2.1 Hz, 1H), 7.84 (dd, J = 8.7, 2.4 Hz, 1H), 6.95 (d,J = 2.4 Hz, 1H), 6.90 (d, J = 2.4 Hz, 1H), 6.37 (d, J = 8.7 Hz, 1H),5.94 (d, J = 3.9 Hz, 1H), 4.92 (d, J = 7.8 Hz, 1H), 4.79 (s, 1H),3.98-3.84 (m, 5H), 3.39- 3.26 (m, 4H), 2.98 (d, J = 4.8 Hz, 3H),2.27-2.12 (m, 2H), 1.97-1.83 (m, 6H) 145

477.2 (400 MHz, CDCl₃) δ 8.69 (d, J = 1.9 Hz, 1H), 8.61 (d, J = 1.9 1H),8.20 (s, 1H), 7.61 (d, J = 8.2 Hz, 1H), 6.95 (d, J = 2.4 Hz, 1H), 6.91(d, J = 2.4 Hz, 1H), 6.43 (d, J = 7.9 Hz, 1H), 4.81 (s, 1H), 3.98-3.81(m, 5H), 3.40-3.27 (m, 4H), 3.09 (s, 6H), 2.27-2.15 (m, 2H), 1.99-1.83(m, 6H) 146

464.17 (CDCl₃) δ 8.76 (d, J = 2.0 Hz, 1H), 8.71 (d, J = 1.9 Hz, 1H),8.63 (d, J = 1.9 Hz, 1H), 7.99 (dd, J = 8.8, 2.2 Hz, 1H), 6.97 (d, J =2.5 Hz, 1H), 6.92 (d, J = 2.5 Hz, 1H), 6.38 (d, J = 8.9 Hz, 1H), 5.13(s, 1H), 4.82 (s, 1H), 4.11-3.73 (m, 8H), 3.44-3.27 (m, 4H), 2.30- 2.17(m, 2H), 2.07-1.79 (m, 6H) 152

464.21 (CDCl₃) δ 8.70 (d, J = 1.9 Hz, 1H), 8.64 (d, J = 1.9 Hz, 1H),7.94 (s, 2H), 6.99-6.90 (m, 2H), 4.82 (s, 1H), 4.05-3.87 (m, 5H), 3.44(q, J = 6.3 Hz, 1H), 3.39-3.26 (m, 4H), 2.23 (d, J = 12.4 Hz, 2H), 2.03-1.82 (m, 6H), 1.23 (d, J = 6.3 Hz, 6H) 155

478.3 (CDCl₃) δ 8.71 (d, J = 1.9 Hz, 1H), 8.64 (d, J = 1.9 Hz, 1H),7.02-6.89 (m, 2H), 4.83 (s, 1H), 4.08-3.87 (m, 5H), 3.44-3.33 (m, 4H),3.27 (d, J = 26.0 Hz, 4H), 2.23 (d, J = 9.8 Hz, 2H), 2.05-1.80 (m, 6H),1.14 (s, 6H) 158

(CDCl₃) δ 8.71 (d, J = 2.0 Hz, 1H), 8.64 (d, J = 1.9 Hz, 1H), 8.41 (s,2H), 6.97 (d, J = 2.4 Hz, 1H), 6.93 (d, J = 2.5 Hz, 1H), 5.32 (d, J =8.1 Hz, 1H), 4.82 (s, 1H), 4.07 (s, 1H), 4.00-3.88 (m, 4H), 3.43-3.29(m, 4H), 2.22 (s, 2H), 2.06- 1.81 (m, 6H) 160

436.2 (400 MHz, CDCl₃) δ 8.69 (d, J = 1.9 Hz, 1H), 8.61 (d, J = 1.9 Hz,1H), 8.05 (d, J = 2.2 Hz, 1H), 7.46 (dd, J = 8.5, 2.3 Hz, 1H), 6.94 (d,J = 2.4 Hz, 1H), 6.90 (d, J = 2.4 Hz, 1H), 6.39 (d, J = 8.6 Hz, 1H),4.77 (s, 1H), 4.53 (s, 2H), 3.95-3.87 (m, 5H), 3.36-3.32 (m, 4H),2.25-2.12 (m, 2H), 1.90 (d, J = 4.4 Hz, 6H) 161

451.28 (CDCl₃) δ 8.69 (d, J = 1.9 Hz, 1H), 8.61 (d, J = 1.9 Hz, 1H),8.26 (s, 2H), 6.95 (d, J = 2.5 Hz, 1H), 6.90 (d, J = 2.6 Hz, 1H), 5.23(d, J = 8.1 Hz, 1H), 4.80 (d, J = 5.8 Hz, 1H), 4.26 (s, 2H), 4.03 (s,1H), 3.97- 3.85 (m, 4H), 3.44-3.21 (m, 7H), 2.32-2.09 (m, 2H), 2.06-1.70(m, 6H) 162

492.29 (DMSO-d₆) δ 8.81-8.64 (m, 3H), 8.58 (d, J = 1.9 Hz, 1H), 7.99 (d,J = 7.6 Hz, 1H), 7.76 (d, J = 7.4 Hz, 1H), 7.14 (d, J = 2.5 Hz, 1H),6.84 (d, J = 2.3 Hz, 1H), 4.92 (s, 1H), 3.98-3.84 (m, 1H), 4.05 (dq, J =13.5, 6.7 Hz, 1H), 3.79 (d, J = 9.6 Hz, 4H), 3.32 (d, J = 8.2 Hz, 4H),2.06 (d, J = 11.8 Hz, 2H), 1.96-1.66 (m, 6H), 1.14 (d, J = 6.6 Hz, 6H)164

484.12 (400 MHz, CDCl₃) δ 8.60 (d, J = 1.9 Hz, 1H), 8.52 (d, J = 1.9 Hz,1H), 8.01 (d, J = 2.1 Hz, 1H), 7.35 (dd, J = 8.8, 2.5 Hz, 1H), 6.86 (d,J = 2.5 Hz, 1H), 6.80 (d, J = 2.5 Hz, 1H), 6.20 (d, J = 8.4 Hz, 1H),4.68 (s, 1H), 4.47 (d, J = 8.0 Hz, 1H), 3.92-3.80 (m, 4H), 3.74 (s, 1H),3.34-3.14 (m, 4H), 2.18-2.02 (m, 2H), 1.94-1.67 (m, 6H) 187

492.26 (CDCl₃) δ 8.70 (d, J = 1.9 Hz, 1H), 8.63 (d, J = 1.9 Hz, 1H),7.70 (s, 1H), 6.96 (q, J = 2.6 Hz, 2H), 5.75 (d, J = 6.5 Hz, 1H), 4.82(s, 1H), 4.65 (s, 1H), 3.93 (dd, J = 5.9, 3.8 Hz, 4H), 3.63 (s, 4H),3.36 (dd, J = 6.1, 3.7 Hz, 4H), 2.14 (s, 2H), 1.95 (d, J = 31.2 Hz, 6H)189

474.147 (400 MHz, CDCl₃) δ 8.69 (d, J = 1.6 Hz, 1H), 8.61 (d, J = 1.7Hz, 1H), 8.32 (s, 1H), 7.56 (d, J = 6.7 Hz, 1H), 6.96 (d, J = 2.2 Hz,1H), 6.90 (d, J = 2.3 Hz, 1H), 6.40 (d, J = 8.7 Hz, 1H), 4.96 (s, 1H),4.80 (s, 1H), 4.01-3.84 (m, 5H), 3.42- 3.24 (m, 4H), 2.21 (d, J = 8.4Hz, 2H), 1.92 (d, J = 6.4 Hz, 6H) 190

(CDCl₃) δ 8.52 (s, 1H), 8.29 (d, J = 4.8 Hz, 2H), 6.90 (d, J = 2.5 Hz,1H), 6.85 (d, J = 2.5 Hz, 1H), 6.52 (t, J = 4.8 Hz, 1H), 5.36 (s, 1H),4.79 (dq, J = 5.6, 2.9 Hz, 1H), 4.04 (dp, J = 8.0, 3.8 Hz, 1H),3.97-3.85 (m, 4H), 3.38-3.26 (m, 4H), 2.70 (s, 3H), 2.29-2.11 (m, 2H),2.00-1.78 (m, 6H) 191

421.24 (CDCl₃) δ 8.52 (s, 1H), 8.29 (d, J = 4.8 Hz, 2H), 6.90 (d, J =2.5 Hz, 1H), 6.85 (d, J = 2.5 Hz, 1H), 6.53 (t, J = 4.8 Hz, 1H), 5.42(d, J = 7.9 Hz, 1H), 4.80 (dq, J = 5.9, 2.9 Hz, 1H), 4.11-3.98 (m, 1H),3.91 (dd, J = 5.9, 3.8 Hz, 4H), 3.42-3.21 (m, 4H), 2.71 (s, 3H),2.31-2.07 (m, 2H), 2.04-1.77 (m, 6H) 193

524.21 (CDCl₃) δ 8.70 (d, J = 1.9 Hz, 1H), 8.63 (d, J = 1.9 Hz, 1H),7.95 (s, 2H), 7.05-6.88 (m, 2H), 4.92 (s, 1H), 4.79 (s, 1H), 3.93 (t, J= 4.8 Hz, 5H), 3.60- 3.44 (m, 4H), 3.37 (d, J = 10.5 Hz, 9H), 2.20 (d, J= 6.9 Hz, 2H), 1.91 (d, J = 4.6 Hz, 6H) 194

437.23 (CDCl₃) δ 8.70 (d, J = 1.9 Hz, 1H), 8.62 (d, J = 1.9 Hz, 1H),8.28 (d, J = 0.9 Hz, 1H), 6.97 (d, J = 2.5 Hz, 1H), 6.91 (d, J = 2.5 Hz,1H), 5.74-5.58 (m, 1H), 5.16 (d, J = 8.0 Hz, 1H), 4.82 (dq, J = 5.4, 2.7Hz, 1H), 4.06-3.85 (m, 7H), 3.70 (d, J = 14.0 Hz, 1H), 3.42-3.28 (m,4H), 2.30-2.16 (m, 2H), 1.99-1.75 (m, 6H) 195

431.22 (CDCl₃) δ 8.70 (d, J = 1.9 Hz, 1H), 8.62 (d, J = 2.0 Hz, 1H),7.43 (dd, J = 8.6, 7.2 Hz, 1H), 7.02-6.86 (M, 3H), 6.55 (dd, J = 8.6,0.8 Hz, 1H), 4.80 (d, J = 6.6 Hz, 2H), 4.10-3.81 (m, 5H), 3.47-3.25 (m,4H), 2.30-2.11 (m, 2H), 1.99-1.81 (m, 6H) 196

463.27 (CDCl₃) δ 8.71 (d, J = 1.9 Hz, 1H), 8.63 (d, J = 1.9 Hz, 1H),7.61 (d, J = 9.4 Hz, 1H), 7.41 (d, J = 7.2 Hz, 1H), 7.05-6.87 (m, 2H),6.65 (s, 1H), 4.86 (s, 1H), 4.04-3.87 (m, 7H), 3.74 (s, 1H), 3.43-3.27(m, 4H), 2.21 (d, J = 10.8 Hz, 2H), 1.98 (d, J = 23.5 Hz, 6H) 197

421.69 (400 MHz, CDCl₃) δ 8.68 (d, J = 1.9 Hz, 1H), 8.61 (d, J = 1.9 Hz,1H), 8.13-8.04 (m, 1H), 7.67 (d, J = 2.4 Hz, 1H), 6.99-6.93 (m, 2H),6.90 (d, J = 2.4 Hz, 1H), 6.33 (d, J = 8.7 Hz, 1H), 4.77 (s, 1H),3.99-3.65 (m, 7H), 3.39-3.27 (m, 4H), 2.25-2.10 (m, 2H), 1.96-1.82 (m,6H) 198

532.11 (400 MHz, CDCl₃) δ 8.69 (d, J = 1.6 Hz, 1H), 8.61 (d, J = 1.7 Hz,1H), 8.57 (d, J = 2.1 Hz, 1H), 7.89 (dd, J = 8.9, 2.3 Hz, 1H), 6.95 (s,1H), 6.90 (s, 1H), 6.62 (d, J = 9.0 Hz, 1H), 5.99 (d, J = 7.7 Hz, 1H),4.78 (s, 1H), 4.18 (s, 1H), 4.03-3.81 (m, 4H), 3.76-3.60 (m, 4H),3.42-3.25 (m, 4H), 2.62-2.44 (m, 4H), 2.36 (s, 3H), 2.29-2.14 (m, 2H),1.99-1.84 (m, 6H) 199

533.01 (400 MHz, CDCl₃) δ 8.73- 8.65 (m, 3H), 8.60 (d, J = 1.9 Hz, 1H),6.95 (d, J = 2.4 Hz, 1H), 6.89 (d, J = 2.4 Hz, 1H), 5.93 (d, J = 8.0 Hz,1H), 4.79 (s, 1H), 4.23-4.09 (m, 1H), 3.97-3.88 (m, 8H), 3.40-3.25 (m,4H), 2.53-2.42 (m, 4H), 2.34 (s, 3H), 2.26-2.18 (m, 2H), 1.97-1.83 (m,6H) 200

549.17 (400 MHz, CDCl₃) δ 8.69 (d, J = 1.9 Hz, 1H), 8.61 (d, J = 1.9 Hz,1H), 7.51-7.42 (m, 2H), 6.98-6.85 (m, 3H), 6.07 (d, J = 8.0 Hz, 1H),4.77 (s, 1H), 4.25-4.11 (m, 1H), 3.99-3.85 (m, 4H), 3.40-3.27 (m, 4H),3.26-3.14 (m, 4H), 2.67-2.52 (m, 4H), 2.36 (s, 3H), 2.28-2.13 (m, 2H),1.98-1.84 (m, 6H) 201

549.1 (400 MHz, CDCl₃) δ 9.47 (s, 1H), 8.68 (d, J = 1.7 Hz, 1H), 8.56(d, J = 1.7 Hz, 1H), 8.21- 8.11 (m, 1H), 6.97-6.85 (m, 4H), 4.82 (s,1H), 4.23-4.07 (m, 1H), 3.96-3.87 (m, 4H), 3.38-3.29 (m, 4H), 3.03 (s,4H), 2.65 (s, 4H), 2.37-2.25 (m, 5H), 2.02-1.84 (m, 6H) 202

464.13 (400 MHz, CDCl₃) δ 8.69 (d, J = 1.8 Hz, 1H), 8.63 (d, J = 1.9 Hz,1H), 8.27 (dd, J = 4.7, 1.9 Hz, 1H), 8.19 (s, 1H), 8.11 (d, J = 6.8 Hz,1H), 6.95 (d, J = 2.4 Hz, 1H), 6.92 (d, J = 2.3 Hz, 1H), 6.51 (dd, J =7.4, 4.9 Hz, 1H), 4.76 (s, 1H), 4.30 (s, 1H), 4.02-3.90 (m, 4H), 3.88(s, 3H), 3.41-3.26 (m, 4H), 2.29-2.11 (m, 2H), 2.11-1.85 (m, 6H) 203

432.58 (CDCl₃) δ 8.70 (d, J = 1.9 Hz, 1H), 8.60 (d, J = 1.9 Hz, 1H),8.15 (s, 1H), 6.93 (dd, J = 17.9, 2.5 Hz, 2H), 6.43 (d, J = 6.1 Hz, 1H),5.20 (s, 1H), 4.82 (s, 1H), 4.00-3.82 (m, 4H), 3.44-3.25 (m, 4H), 2.23(d, J = 11.2 Hz, 2H), 1.91 (s, 6H) 204

421.65 (CDCl₃) δ 8.70 (d, J = 1.9 Hz, 1H), 8.61 (d, J = 1.9 Hz, 1H),8.09 (d, J = 6.0 Hz, 1H), 6.93 (dd, J = 16.1, 2.5 Hz, 2H), 6.15 8.0 Hz,1H), 4.81 (td, J = 5.5, 2.7 Hz, 1H), 3.97- 3.87 (m, 4H), 3.49 (s, 1H),3.39-3.27 (m, 4H), 2.49 (s, 3H), 2.27-2.14 (m, 2H), 2.03-1.80 (m, 6H)205

436.63 (CDCl₃) δ 8.70 (d, J = 1.9 Hz, 1H), 8.61 (dd, J = 5.3, 1.9 Hz,1H), 8.19-8.06 (m, 1H), 6.99- 6.84 (m, 2H), 5.34-5.21 (m, 1H), 4.76 (d,J = 9.7 Hz, 3H), 3.92 (t, J = 4.9 Hz, 4H), 3.81 (s, 1H), 3.33 (dd, J =5.7, 4.1 Hz, 4H), 2.87 (d, J = 5.2 Hz, 3H), 2.19 (d, J = 8.5 Hz, 2H),1.88 (dd, J = 13.3, 5.1 Hz, 6H) 206

436.18 (400 MHz, CDCl₃) δ 8.68 (d, J = 1.9 Hz, 1H), 8.61 (d, J = 1.9 Hz,1H), 7.82 (d, J = 2.8 Hz, 1H), 7.09 (dd, J = 8.9, 3.0 Hz, 1H), 6.94 (d,J = 2.5 Hz, 1H), 6.90 (d, J = 2.5 Hz, 1H), 6.37 (d, J = 8.8 Hz, 1H),4.77 (s, 1H), 4.29 (bs, 1H), 3.98-3.87 (m, 4H), 3.85-3.79 (m, 1H), 3.77(s, 3H), 3.41- 3.24 (m, 4H), 2.27-2.12 (m, 2H), 1.97-1.79 (m, 6H) 207

(CDCl₃) δ 8.71 (d, J = 2.0 Hz, 1H), 8.64 (d, J = 1.9 Hz, 1H), 8.44 (s,1H), 6.98 (d, J = 2.5 Hz, 1H), 6.92 (d, J = 2.5 Hz, 1H), 6.82 (d, J =4.7 Hz, 1H), 5.56 (d, J = 30.8 Hz, 1H), 4.83 (d, J = 5.2 Hz, 1H), 4.04(s, 2H), 3.97-3.84 (m, 4H), 3.44- 3.29 (m, 4H), 2.23 (d, J = 8.2 Hz,2H), 2.02-1.77 (m, 6H) 208

435.6 (CDCl₃) δ 8.70 (d, J = 1.9 Hz, 1H), 8.64 (d, J = 1.9 Hz, 1H), 8.16(s, 2H), 7.00-6.84 (m, 2H), 5.32 (s, 1H), 4.81 (s, 1H), 4.03 (s, 1H),3.96-3.85 (m, 4H), 3.43-3.32 (m, 4H), 2.49 (q, J = 7.6 Hz, 2H), 2.21 (d,J = 8.8 Hz, 2H), 2.06-1.75 (m, 6H), 1.21 (t, J = 7.6 Hz, 3H) 209

(CDCl₃) δ 8.70 (d, J = 1.9 Hz, 1H), 8.63 (d, J = 1.9 Hz, 1H), 8.18 (s,2H), 7.01-6.88 (m, 2H), 5.30 (d, J = 9.0 Hz, 1H), 4.81 (d, J = 5.9 Hz,1H), 4.02 (s, 1H), 3.96-3.85 (m, 4H), 3.42-3.29 (m, 4H), 2.78 (p, J =6.9 Hz, 1H), 2.31-2.14 (m, 2H), 2.01-1.83 (m, 6H), 1.25 (d, J = 6.9 Hz,6H) 210

450.17 (400 MHz, methanol-d₄) δ 8.69 (d, J = 2.0 Hz, 1H), 8.57 (d, J =2.0 Hz, 1H), 7.92 (d, J = 6.2 Hz, 1H), 7.17-7.10 (m, 2H), 7.02 (d, J =7.1 Hz, 1H), 6.90 (d, J = 2.5 Hz, 1H), 4.93 (s, 1H), 3.93-3.87 (m, 4H),3.84-3.79 (m, 1H), 3.44-3.37 (m, 4H), 2.24-2.15 (m, 2H), 1.97-1.82 (m,6H) 215

451.21 (CDCl₃) δ 8.70 (d, J = 1.9 Hz, 1H), 8.61 (d, J = 1.9 Hz, 1H),8.25 (d, J = 0.9 Hz, 1H), 6.93 (dd, J = 17.1, 2.5 Hz, 2H), 5.64 (d, J =0.9 Hz, 1H), 5.00 (d, J = 8.1 Hz, 1H), 4.80 (dq, J = 5.5, 2.7 Hz, 1H),4.33 (q, J = 7.1 Hz, 2H), 3.97-3.87 (m, 4H), 3.71 (s, 1H), 3.38-3.29 (m,4H), 2.27-2.14 (m, 2H), 2.03-1.80 (m, 6H), 1.37 (t, J = 7.1 Hz, 3H) 219

471.06 (CDCl₃) δ 8.70 (d, J = 1.9 Hz, 1H), 8.61 (d, J = 1.9 Hz, 1H),6.96 (d, J = 2.5 Hz, 1H), 6.89 (d, J = 2.5 Hz, 1H), 6.03 (s, 1H), 5.00(s, 1H), 4.79 (s, 1H), 3.92 (d, J = 9.3 Hz, 7H), 3.41- 3.25 (m, 4H),2.28-2.13 (m, 2H), 1.92 (d, J = 18.6 Hz, 6H) 223

435.18 (CDCl₃) δ 8.71 (d, J = 1.9 Hz, 1H), 8.61 (d, J = 1.9 Hz, 1H),6.94 (dd, J = 15.2, 2.5 Hz, 2H), 6.07 (s, 1H), 4.82 (dt, J = 5.8, 3.0Hz, 1H), 4.02- 3.83 (m, 4H), 3.77-3.57 (m, 1H), 3.43-3.28 (m, 4H), 2.52(s, 3H), 2.37 (s, 3H), 2.30- 2.16 (m, 2H), 2.05-1.78 (m, 6H) 224

475.02 (CDCl₃) δ 8.72 (d, J = 1.9 Hz, 1H), 8.63 (d, J = 1.9 Hz, 1H),8.27 (s, 1H), 6.98 (d, J = 2.4 Hz, 1H), 6.92 (d, J = 2.6 Hz, 1H), 6.44(d, J = 6.0 Hz, 1H), 4.84 (s, 1H), 3.93 (dd, J = 6.0, 3.7 Hz, 4H),3.43-3.25 (m, 4H), 2.23 (s, 2H), 2.08-1.83 (m, 6H) 225

451.16 (CDCl₃) δ 8.70 (d, J = 1.9 Hz, 1H), 8.62 (d, J = 1.9 Hz, 1H),8.49 (d, J = 1.1 Hz, 1H), 6.93 (dd, J = 16.7, 2.5 Hz, 2H), 6.45 (d, J =1.2 Hz, 1H), 5.01 (s, 1H), 4.81 (s, 1H), 4.39 (d, J = 0.9 Hz, 2H),3.97-3.87 (m, 4H), 3.49 (s, 3H), 3.39- 3.29 (m, 4H), 2.22 (d, J = 9.4Hz, 2H), 1.99-1.87 (m, 6H) 226

463.18 (CDCl₃) δ 8.70 (d, J = 2.0 Hz, 1H), 8.63 (d, J = 1.9 Hz, 1H),6.95 (d, J = 2.5 Hz, 1H), 6.91 (d, J = 2.5 Hz, 1H), 4.75 (d, J = 5.6 Hz,1H), 4.66 (s, 1H), 4.32 (s, 1H), 4.00-3.83 (m, 4H), 3.43-3.22 (m, 4H),2.46 (d, J = 15.1 Hz, 5H), 2.36 (s, 3H), 2.19 (q, J = 6.3, 3.9 Hz, 2H),1.12 (t, J = 7.6 Hz, 3H) 227

453.2 (CDCl₃) δ 8.72 (d, J = 1.9 Hz, 1H), 8.64 (d, J = 1.9 Hz, 1H), 8.33(d, J = 1.8 Hz, 1H), 7.04- 6.87 (m, 2H), 5.15 (s, 1H), 4.82 (dq, J =5.2, 2.6 Hz, 1H), 4.24 (dt, J = 8.3, 4.7 Hz, 1H), 4.03-3.86 (m, 4H),3.44-3.28 (m, 4H), 2.74 (qd, J = 7.6, 2.3 Hz, 2H), 2.24 (dq, J = 9.6,4.6 Hz, 2H), 2.09-1.85 (m, 6H), 1.29 (t, J = 7.6 Hz, 3H) 233

451.2 (CDCl₃) δ 8.70 (d, J = 1.9 Hz, 1H), 8.63 (d, J = 1.9 Hz, 1H), 8.06(s, 2H), 7.01-6.86 (m, 2H), 5.00 (d, J = 8.1 Hz, 1H), 4.80 (d, J = 5.6Hz, 1H), 4.10-3.82 (m, 7H), 3.42-3.26 (m, 5H), 2.20 (d, J = 8.2 Hz, 2H),2.01-1.80 (m, 6H), 1.39 (t, J = 7.0 Hz, 3H) 234

432.17 (CDCl₃) δ 8.72 (d, J = 1.9 Hz, 1H), 8.60 (t, J = 1.9 Hz, 2H),6.97 (d, J = 2.5 Hz, 1H), 6.91 (d, J = 2.5 Hz, 1H), 6.70 (d, J = 1.2 Hz,1H), 4.85 (d, J = 4.9 Hz, 1H), 3.98-3.84 (m, 4H), 3.42-3.26 (m, 4H),2.33-2.17 (m, 2H), 2.01-1.77 (m, 5H) 235

505.04 (CDCl₃) δ 8.68 (d, J = 2.0 Hz, 1H), 8.59 (d, J = 1.9 Hz, 1H),8.36 (s, 2H), 6.97-6.86 (m, 2H), 5.53 (d, J = 8.1 Hz, 1H), 4.96-4.72 (m,3H), 4.09-3.86 (m, 5H), 3.33 (dd, J = 5.8, 4.0 Hz, 4H), 2.28-2.10 (m,2H), 1.98-1.78 (m, 6H) 236

505.17 (CDCl₃) δ 8.68 (d, J = 2.0 Hz, 1H), 8.59 (d, J = 1.9 Hz, 1H),8.36 (s, 2H), 6.97-6.86 (m, 2H), 5.53 (d, J = 8.1 Hz, 1H), 4.96-4.72 (m,3H), 4.09-3.86 (m, 5H), 3.33 (dd, J = 5.8, 4.0 Hz, 4H), 2.28-2.10 (m,2H), 1.98-1.78 (m, 6H) 237

451.16 (CDCl₃) δ 8.69 (d, J = 1.9 Hz, 1H), 8.61 (d, J = 1.9 Hz, 1H),8.31 (s, 2H), 6.99-6.87 (m, 2H), 5.34 (d, J = 8.1 Hz, 1H), 4.87-4.73 (m,2H), 4.06-3.87 (m, 6H), 3.38-3.29 (m, 4H), 2.20 (q, J = 5.8 Hz, 2H),2.04-1.84 (m, 6H), 1.51 (d, J = 6.5 Hz, 3H) 241

445.54 (400 MHz, CDCl₃) δ 8.69 (d, J = 1.9 Hz, 1H), 8.61 (d, J = 1.9 Hz,1H), 7.52 (d, J = 8.7 Hz, 1H), 6.96 (d, J = 2.5 Hz, 1H), 6.90 (d, J =2.5 Hz, 1H), 6.23 (d, J = 8.7 Hz, 1H), 5.09 (s, 1H), 4.80 (s, 1H), 4.00-3.78 (m, 5H), 3.40-3.24 (m, 4H), 2.56 (s, 3H), 2.29-2.14 (m, 2H),2.01-1.80 (m, 6H) 242

445.54 (400 MHz, CDCl₃) δ 8.70 (d, J = 1.8 Hz, 1H), 8.62 (d, J = 1.9 Hz,1H), 8.30 (d, J = 1.8 Hz, 1H), 7.37 (s, 1H), 6.96 (d, J = 2.3 Hz, 1H),6.91 (d, J = 2.3 Hz, 1H), 4.79 (s, 1H), 4.74 (s, 1H), 4.28 (s, 1H),4.01-3.83 (m, 4H), 3.40-3.25 (m, 4H), 2.30-2.17 (m, 2H), 2.11 (s, 3H),1.99-1.87 (m, 6H) 243

465.2 (CDCl₃) δ 8.69 (d, J = 2.0 Hz, 1H), 8.62 (d, J = 1.9 Hz, 1H), 5.36(s, 1H), 4.03 (s, 1H), 1.57 (s, 6H), 2.02-1.80 (m, 6H), 8.42 (s, 2H),6.99-6.87 (m, 2H), 4.80 (s, 1H), 3.92 (dd, J = 6.0, 3.7 Hz, 4H),3.39-3.29 (m, 4H), 2.20 (d, J = 9.0 Hz, 2H) 245

467.14 (CDCl₃) δ 8.61 (d, J = 1.9 Hz, 1H), 8.53 (d, J = 1.9 Hz, 1H),6.84 (dd, J = 18.2, 2.5 Hz, 2H), 5.26 (s, 1H), 4.78 (d, J = 8.0 Hz, 1H),4.68 (d, J = 5.6 Hz, 1H), 3.82 (t, J = 4.0 Hz, 10H), 3.30-3.15 (m, 4H),2.10 (q, J = 6.2, 5.7 Hz, 2H), 1.94-1.69 (m, 6H) 246

421.23 (CDCl₃) δ 8.61 (d, J = 1.9 Hz, 1H), 8.54 (d, J = 1.9 Hz, 1H),8.06 (d, J = 5.0 Hz, 1H), 6.94- 6.78 (m, 2H), 6.32 (d, J = 5.0 Hz, 1H),5.09 (s, 1H), 4.70 (d, J = 6.0 Hz, 1H), 3.95 (s, 1H), 3.91-3.77 (m, 5H),3.37-3.13 (m, 4H), 2.20-2.00 (m, 2H), 1.94-1.71 (m, 6H) 247

465.1 (CDCl₃) δ 8.71 (d, J = 1.9 Hz, 1H), 8.64 (t, J = 2.4 Hz, 1H), 8.05(s, 2H), 7.04-6.87 (m, 2H), 5.20 (s, 1H), 4.80 (s, 1H), 4.30 (p, J = 6.1Hz, 1H), 4.05-3.81 (m, 4H), 3.44-3.27 (m, 4H), 2.22 (t, J = 7.3 Hz, 2H),1.93 (d, J = 4.6 Hz, 6H), 1.33 (d, J = 6.1 Hz, 6H) 254

406.57 (400 MHz, CDCl₃) δ 8.71 (d, J = 1.9 Hz, 1H), 8.63 (d, J = 1.9 Hz,1H), 8.15-8.04 (m, 1H), 7.46-7.39 (m, 1H), 6.97 (d, J = 2.5 Hz, 1H),6.93 (d, J = 2.5 Hz, 1H), 6.60-6.52 (m, 1H), 6.41 (d, J = 8.4 Hz, 1H),4.80 (s, 1H), 4.68 (s, 1H), 3.98-3.85 (m, 5H), 3.41- 3.30 (m, 4H),2.27-2.15 (m, 2H), 1.97- 1.86 (m, 6H) 256

420.1 (CDCl₃) δ 8.71 (d, J = 1.9 Hz, 1H), 8.64 (d, J = 1.9 Hz, 1H), 7.80(d, J = 1.5 Hz, 1H), 7.47 (d, J = 1.5 Hz, 1H), 7.02-6.86 (m, 2H), 4.80(s, 1H), 4.27 (d, J = 8.3 Hz, 1H), 4.00-3.72 (m, 7H), 3.35 (dd, J = 6.0,3.8 Hz, 4H), 2.23 (s, 2H), 2.02-1.80 (m, 6H) 257

437.1 (CDCl₃) δ 8.71 (d, J = 1.9 Hz, 1H), 8.63 (dd, J = 1.9, 0.7 Hz,1H), 7.99-7.88 (m, 1H), 7.02- 6.87 (m, 2H), 6.34 (d, J = 8.4 Hz, 1H),4.78 (s, 1H), 4.40 (d, J = 8.2 Hz, 1H), 4.03-3.75 (m, 5H), 3.35 (dd, J =6.0, 3.7 Hz, 4H), 2.18 (s, 5H), 2.03-1.81 (m, 6H) 260

437.24 (CDCl₃) δ 8.70 (d, J = 1.9 Hz, 1H), 8.62 (d, J = 1.9 Hz, 1H),6.93 (dd, J = 14.5, 2.5 Hz, 2H), 6.79 (d, J = 9.4 Hz, 1H), 6.64 (d, J =9.4 Hz, 1H), 4.80 (d, J = 5.5 Hz, 1H), 4.27 (d, J = 7.5 Hz, 1H), 4.14(s, 1H), 4.02 (s, 3H), 3.98-3.88 (m, 4H), 3.42-3.29 (m, 4H), 2.31-2.12(m, 2H), 1.95 (ddd, J = 17.2, 9.2, 6.0 Hz, 6H) 263

480.22 (CDCl₃) δ 8.70 (d, J = 1.9 Hz, 1H), 8.61 (d, J = 1.9 Hz, 1H),8.11 (d, J = 0.9 Hz, 1H), 6.93 (dd, J = 15.5, 2.5 Hz, 2H), 5.30 (d, J =1.0 Hz, 1H), 5.16 (s, 1H), 4.95 (s, 1H), −0.17-−0.23 (m, 0H), 4.80 (s,1H), 3.92 (dd, J = 5.9, 3.7 Hz, 4H), 3.74 (s, 1H), 3.57 (dd, J = 5.6,4.6 Hz, 2H), 3.50-3.29 (m, 9H), 2.20 (d, J = 9.1 Hz, 2H), 1.93 (d, J =13.1 Hz, 6H) 264

494.1 (CDCl₃) δ 8.69 (d, J = 1.9 Hz, 1H), 8.62 (d, J = 2.0 Hz, 1H), 7.76(d, J = 5.8 Hz, 1H), 7.02- 6.86 (m, 2H), 5.66 (d, J = 5.9 Hz, 1H), 4.80(d, J = 5.5 Hz, 1H), 4.63 (s, 1H), 3.97-3.84 (m, 5H), 1.30-1.20 (m, 1H),3.64 (s, 2H), 3.38-3.28 (m, 4H), 1.37 (s, 6H), 5.16-4.86 (m, 1H),2.24-2.13 (m, 2H), 1.96-1.82 (m, 6H) 265

421.18 (CDCl₃) δ 8.70 (d, J = 1.9 Hz, 1H), 8.61 (d, J = 1.9 Hz, 1H),8.47 (d, J = 1.1 Hz, 1H), 6.93 (dd, J = 16.7, 2.5 Hz, 2H), 6.17 (t, J =0.9 Hz, 1H), 4.95 (s, 1H), 4.81 (td, J = 5.3, 2.5 Hz, 1H), 4.07-3.83 (m,5H), 3.39-3.29 (m, 4H), 2.34 (s, 3H), 2.21 (dt, J = 11.1, 5.1 Hz, 2H),2.04-1.76 (m, 6H) 267

447.11 (CDCl₃) δ 8.70 (d, J = 1.9 Hz, 1H), 8.60 (d, J = 1.9 Hz, 1H),8.03 (d, J = 6.1 Hz, 1H), 6.93 (dd, J = 18.6, 2.5 Hz, 2H), 6.13 (d, J =6.1 Hz, 1H), 5.09 (s, 1H), 4.79 (s, 1H), 4.03- 3.78 (m, 5H), 3.34 (dd, J= 6.0, 3.8 Hz, 4H), 2.20 (d, J = 8.1 Hz, 2H), 1.96 (s, 7H), 1.10 (d, J =2.8 Hz, 2H), 1.00 (d, J = 8.0 Hz, 2H) 268

437.19 (CDCl₃) δ 8.69 (d, J = 1.9 Hz, 1H), 8.60 (d, J = 1.9 Hz, 1H),7.95 (d, J = 5.8 Hz, 1H), 6.92 (dd, J = 17.9, 2.5 Hz, 2H), 5.99 (d, J =5.9 Hz, 1H), 5.01 (s, 1H), 4.79 (dt, J = 6.9, 3.4 Hz, 1H), 3.91 (d, J =8.7 Hz, 8H), 3.43-3.25 (m, 4H), 2.32- 2.09 (m, 2H), 2.05-1.74 (m, 6H)269

468.13 (CDCl₃) δ 8.69 (d, J = 1.9 Hz, 1H), 8.62 (d, J = 1.9 Hz, 1H),7.70 (d, J = 6.8 Hz, 1H), 6.92 (dd, J = 14.4, 2.5 Hz, 2H), 4.86 (d, J =8.1 Hz, 1H), 4.75 (dt, J = 8.6, 4.0 Hz, 1H), 3.97- 3.87 (m, 5H),3.38-3.28 (m, 4H), 3.14 (d, J = 2.2 Hz, 6H), 2.24-2.07 (m, 2H),2.02-1.79 (m, 6H) 272

406.53 (CDCl₃) δ 8.69 (d, J = 1.9 Hz, 1H), 8.61 (d, J = 1.9 Hz, 1H),8.04 (d, J = 2.8 Hz, 1H), 7.94 (dd, J = 4.7, 1.3 Hz, 1H), 7.11 (dd, J =8.3, 4.7 Hz, 1H), 7.00-6.84 (m, 3H), 4.78 (s, 1H), 3.98-3.88 (m, 4H),3.85 (d, J = 8.1 Hz, 1H), 3.38-3.28 (m, 4H), 2.27-2.15 (m, 2H),1.97-1.84(m, 6H) 273

406.57 (CDCl₃) δ 8.70 (d, J = 1.9 Hz, 1H), 8.61 (d, J = 1.9 Hz, 1H),8.18 (d, J = 6.3 Hz, 2H), 6.96 (d, J = 2.4 Hz, 1H), 6.90 (d, J = 2.4 Hz,1H), 6.51-6.42 (m, 2H), 4.79 (s, 1H), 4.47 (d, J = 7.7 Hz, 1H), 3.98-3.86 (m, 4H), 3.56 (s, 1H), 3.39-3.26 (m, 4H), 2.27-2.15 (m, 2H),2.01-1.79 (m, 6H) 275

519.2 (CDCl₃) δ 8.62 (d, J = 1.9 Hz, 1H), 8.54 (d, J = 1.9 Hz, 1H),6.93-6.77 (m, 2H), 5.20 (s, 1H), 4.78-4.60 (m, 2H), 3.92- 3.79 (m, 5H),3.64 (s, 0H), 3.51 (t, J = 5.1 Hz, 4H), 3.36- 3.16 (m, 4H), 2.40 (t, J =5.1 Hz, 4H), 2.27 (d, J = 4.8 Hz, 6H), 2.16-2.02 (m, 2H), 1.81 (q, J =8.1, 5.7 Hz, 6H) 276

451.53 (CDCl₃) δ 8.69 (d, J = 1.8 Hz, 1H), 8.61 (d, J = 1.9 Hz, 1H),6.94 (d, J = 2.3 Hz, 1H), 6.90 (d, J = 2.3 Hz, 1H), 5.87 (s, 1H), 5.13(s, 1H), 4.76 (s, 1H), 4.04 (s, 1H), 3.98-3.89 (m, 4H), 3.87 (s, 3H),3.41- 3.23 (m, 4H), 2.25 (s, 3H), 2.23-2.09 (m, 2H), 1.99-1.80 (m, 6H)277

459.08 (CDCl₃) δ 8.71 (d, J = 1.9 Hz, 1H), 8.62 (d, J = 1.9 Hz, 1H),8.17 (d, J = 0.9 Hz, 1H), 6.94 (dd, J = 15.8, 2.5 Hz, 2H), 5.31 (s, 1H),4.83 (dp, J = 4.6, 2.5 Hz, 1H), 4.22 (qt, J = 8.5, 4.9 Hz, 1H), 4.03-3.87 (m, 4H), 3.39-3.29 (m, 4H), 2.25 (td, J = 7.9, 6.6, 3.9 Hz, 2H),2.09-1.80 (m, 6H) 282

421.2 (CDCl₃) δ 8.71 (d, J = 1.9 Hz, 1H), 8.63 (d, J = 1.9 Hz, 1H),7.93-7.74 (m, 2H), 7.04-6.82 (m, 2H), 4.80 (s, 1H), 4.50 (d, J = 8.1 Hz,1H), 3.93 (dd, J = 6.4, 3.4 Hz, 5H), 3.41-3.25 (m, 4H), 2.51-2.33 (m,3H), 2.23 (s, 2H), 2.04-1.82 (m, 6H) 283

420.2 (CDCl₃) δ 6 8.62 (d, J = 1.9 Hz, 1H), 8.55 (d, J = 1.9 Hz, 1H),7.88 (dd, J = 5.3, 0.7 Hz, 1H), 6.95-6.79 (m, 2H), 6.32 (ddd, J = 5.2,1.5, 0.7 Hz, 1H), 6.12 (dt, J = 1.6, 0.8 Hz, 1H), 4.71 (d, J = 5.9 Hz,1H), 4.40 (d, J = 8.2 Hz, 1H), 3.98-3.67 (m, 4H), 3.35-3.17 (m, 4H),2.15 (s, 4H), 1.83 (d, J = 5.2 Hz, 5H) 285

420.57 (400 MHz, CDCl₃) δ 8.69 (d, J = 1.9 Hz, 1H), 8.61 (d, J = 1.9 Hz,1H), 8.08 (d, J = 5.7 Hz, 1H), 6.96 (d, J = 2.4 Hz, 1H), 6.90 (d, J =2.4 Hz, 1H), 6.30 (s, 1H), 6.28 (dd, J = 5.7, 2.2 Hz, 1H), 4.78 (s, 1H),4.19 (d, J = 7.7 Hz, 1H), 3.97-3.82 (m, 4H), 3.53 (s, 1H), 3.40- 3.24(m, 4H), 2.41 (s, 3H), 2.27-2.11 (m, 2H), 1.92-1.84 (m, 6H) 286

434.56 (400 MHz, CDCl₃) δ 8.69 (d, J = 1.9 Hz, 1H), 8.60 (d, J = 1.9 Hz,1H), 6.95 (d, J = 2.4 Hz, 1H), 6.89 (d, J = 2.4 Hz, 1H), 6.15 (s, 2H),4.77 (s, 1H), 4.12 (d, J = 7.8 Hz, 1H), 3.99- 3.81 (m, 4H), 3.53 (s,1H), 3.40-3.23 (m, 4H), 2.36 (d, J = 14.5 Hz, 6H), 2.25-2.14 (m, 2H),1.89 (t, J = 7.8 Hz, 6H)

TABLE 2 Cmpd. ESMS No. Compound Structure (M + H) ¹H NMR 287

478.3 1H NMR (300 MHz, Chloroform-d) δ 8.70 (d, J = 1.9 Hz, 1H), 8.62(d, J = 1.9 Hz, 1H), 8.33 (s, 1H), 6.94 (dd, J = 14.3, 2.5 Hz, 2H), 5.80(d, J = 4.9 Hz, 1H), 5.42 (d, J = 8.1 Hz, 1H), 4.88- 4.74 (m, 1H), 4.08(s, 2H), 3.99-3.86 (m, 4H), 3.50 (s, 3H), 3.42- 3.28 (m, 4H), 3.01 (dd,J = 18.4, 5.0 Hz, 3H), 2.54 (s, 3H), 2.28- 2.08 (m, 2H), 2.03- 1.79 (m,6H). 291

419.23 [1] 1H NMR (400 MHz, Chloroform-d) δ 8.42 (d, J = 1.9 Hz, 1H),8.30 (d, J = 1.9 Hz, 1H), 8.04 (d, J = 4.8 Hz, 2H), 6.65-6.56 (m, 2H),6.28 (t, J = 4.8 Hz, 1H), 4.99 (d, J = 8.1 Hz, 1H), 4.60 (d, J = 6.5 Hz,3H), 3.79 (dd, J = 8.2, 4.0 Hz, 0H), 3.62-3.38 (m, 4H), 3.17-3.03 (m,1H), 2.07-1.90 (m, 2H), 1.89-1.59 (m, 7H). 294

410.35 1H NMR (300 MHz, Chloroform-d) δ 8.10 (d, J = 6.0 Hz, 1H),7.36-7.28 (m, 1H), 6.65 (d, J = 2.0 Hz, 1H), 6.13 (d, J = 6.0 Hz, 1H),4.81 (d, J = 8.0 Hz, 1H), 4.00- 3.80 (m, 6H), 3.80- 3.56 (m, 1H), 3.29(dd, J = 5.8, 3.8 Hz, 4H), 3.02-2.84 (m, 2H), 2.48 (s, 3H), 2.36 (td, J= 11.4, 10.9, 2.5 Hz, 2H), 2.14-1.98 (m, 2H), 1.70-1.46 (m, 2H). 295

450.2 1H NMR (300 MHz, Chloroform-d) δ 8.98- 8.78 (m, 2H), 8.33 (d, J =1.9 Hz, 1H), 7.67 (d, J = 1.8 Hz, 1H), 7.30 (d, J = 1.8 Hz, 1H),6.49-6.31 (m, 1H), 5.07 (d, J = 7.6 Hz, 1H), 4.87 (d, J = 5.5 Hz, 1H),4.43 (q, J = 2.8 Hz, 2H), 4.24 (s, 1H), 4.02 (t, J = 5.4 Hz, 2H), 2.71(dtd, J = 13.1, 7.7, 2.7 Hz, 4H), 2.26 (dt, J = 10.4, 5.3 Hz, 2H), 1.97(d, J = 5.3 Hz, 6H), 1.28 (t, J = 7.6 Hz, 3H). 297

447 1H NMR (400 MHz, CDCl3) δ 8.60 (d, J = 1.9 Hz, 1H), 8.49 (d, J = 1.9Hz, 1H), 8.02 (d, J = 6.0 Hz, 1H), 6.78 (dd, J = 10.1, 2.5 Hz, 2H), 6.08(d, J = 6.0 Hz, 1H), 4.96 (s, 1H), 4.74 (d, J = 2.4 Hz, 1H), 4.49 (d, J= 2.3 Hz, 2H), 3.41 (t, J = 5.4 Hz, 2H), 3.12 (dd, J = 11.6, 2.5 Hz,2H), 2.43 (s, 3H), 2.14 (dd, J = 9.6, 4.9 Hz, 2H), 1.98-1.68 (m, 10H).298

459.4 1H NMR (300 MHz, Chloroform-d) δ 8.71 (d, J = 1.9 Hz, 1H), 8.65(d, J = 1.9 Hz, 1H), 7.53 (dt, J = 8.1, 1.0 Hz, 1H), 7.35 (ddd, J = 8.1,6.8, 1.1 Hz, 1H), 7.20 (dt, J = 8.5, 0.9 Hz, 1H), 7.07- 6.89 (m, 3H),4.79 (td, J = 6.1, 3.1 Hz, 1H), 4.02-3.91 (m, 4H), 3.87 (s, 3H), 3.43-3.25 (m, 4H), 2.34- 2.15 (m, 2H), 2.10- 1.88 (m, 6H). 308

478.64 1H NMR (300 MHz, Chloroform-d) δ 8.71 (d, J = 1.9 Hz, 1H), 8.64(d, J = 1.9 Hz, 1H), 7.79 (s, 1H), 7.23 (s, 1H), 6.95 (dd, J = 14.0, 2.5Hz, 2H), 5.23 (d, J = 8.1 Hz, 1H), 4.80 (s, 1H), 4.09 (s, 1H), 3.94 (dd,J = 6.1, 3.6 Hz, 4H), 3.43- 3.23 (m, 4H), 3.02 (d, J = 5.1 Hz, 3H), 2.43(s, 3H), 2.31-2.12 (m, 2H), 1.95 (p, J = 10.0 Hz, 6H). 309

477.54 1H NMR (300 MHz, Chloroform-d) δ 8.68 (d, J = 1.9 Hz, 1H), 8.54(d, J = 1.9 Hz, 1H), 6.85 (s, 2H), 6.30 (s, 1H), 5.06 (d, J = 16.2 Hz,1H), 4.85 (d, J = 6.5 Hz, 3H), 4.44- 4.30 (m, 2H), 4.14 (q, J = 7.1 Hz,1H), 3.89- 3.60 (m, 5H), 3.50 (s, 3H), 3.35 (q, J = 7.2 Hz, 1H), 2.49(s, 3H), 2.24 (d, J = 8.7 Hz, 2H), 2.08 (d, J = 7.7 Hz, 2H), 1.92 (q, J= 9.6, 6.9 Hz, 6H), 1.28 (t, J = 7.1 Hz, 2H). 312

437.33 1H NMR (300 MHz, CDCl3) δ 8.71 (d, J = 1.9 Hz, 1H), 8.65 (d, J =1.9 Hz, 1H), 7.57 (d, J = 3.2 Hz, 1H), 7.35- 7.28 (m, 2H), 6.97 (d, J =2.4 Hz, 1H), 6.91 (d, J = 2.4 Hz, 1H), 5.23 (s, 1H), 4.76 (s, 1H), 4.20(s, 1H), 4.01 (s, 3H), 3.98-3.84 (m, 4H), 3.44-3.29 (m, 4H), 2.27-2.14(m, 2H), 2.05-1.85 (m, 6H). 314

435.32 1H NMR (300 MHz, CDCl3) δ 8.71 (d, J = 1.9 Hz, 1H), 8.63 (d, J =1.9 Hz, 1H), 7.66 (s, 1H), 6.94 (dd, J = 14.9, 2.4 Hz, 2H), 4.84- 4.74(m, 1H), 4.47 (s, 1H), 4.02-3.87 (m, 4H), 3.87-3.75 (m, 1H), 3.47-3.21(m, 4H), 2.38 (d, J = 4.0 Hz, 6H), 2.26-2.13 (m, 2H), 2.01-1.78 (m, 6H).315

476.23 1H NMR (300 MHz, Chloroform-d) δ 8.86 (d, J = 1.9 Hz, 1H),8.59-8.46 (m, 2H), 7.98 (s, 1H), 7.18 (s, 1H), 6.85 (s, 2H), 4.85 (d, J= 6.6 Hz, 2H), 3.90-3.62 (m, 5H), 3.35 (d, J = 8.2 Hz, 1H), 3.03 (d, J =5.1 Hz, 3H), 2.27 (s, 2H), 2.13-1.80 (m, 7H), 10.58 (s, 2H) 316

411.34 1H NMR (300 MHz, Chloroform-d) δ 8.14 (d, J = 6.2 Hz, 1H), 6.50(d, J = 1.7 Hz, 1H), 6.43 (d, J = 1.6 Hz, 1H), 6.21 (d, J = 6.2 Hz, 1H),4.98 (s, 1H), 4.01-3.81 (m, 5H), 3.37-3.18 (m, 5H), 2.54 (s, 3H), 2.20(d, J = 9.1 Hz, 3H), 2.05-1.71 (m, 6H). 317

476.55 1H NMR (300 MHz, Chloroform-d) δ 8.68 (d, J = 1.9 Hz, 1H),8.62-8.41 (m, 2H), 7.79 (s, 1H), 7.33 (d, J = 4.9 Hz, 1H), 6.85 (q, J =2.6 Hz, 3H), 4.85 (d, J = 6.3 Hz, 4H), 4.08 (s, 0H), 3.88- 3.58 (m, 5H),3.35 (q, J = 6.8 Hz, 1H), 3.03 (d, J = 5.1 Hz, 3H), 2.36-2.13 (m, 2H),2.15-1.84 (m, 6H). 318

465.3 1H NMR (300 MHz, Chloroform-d) δ 8.68 (d, J = 1.9 Hz, 1H), 8.56(d, J = 1.9 Hz, 1H), 8.33 (d, J = 1.9 Hz, 1H), 6.85 (s, 2H), 5.07 (d, J= 8.1 Hz, 1H), 4.86 (d, J = 6.4 Hz, 4H), 4.23 (s, 2H), 3.90-3.62 (m,4H), 3.35 (q, J = 7.1 Hz, 1H), 2.73 (qd, J = 7.6, 2.3 Hz, 2H), 2.27 (d,J = 10.1 Hz, 3H), 2.12- 1.82 (m, 6H), 1.28 (t, J = 7.6 Hz, 3H). 319

461.38 1H NMR (300 MHz, Chloroform-d) δ 8.83 (dt, J = 6.4, 1.4 Hz, 2H),8.51 (d, J = 4.7 Hz, 1H), 7.78 (s, 1H), 7.68 (d, J = 1.7 Hz, 1H), 7.34(dd, J = 4.9, 0.9 Hz, 1H), 6.38 (s, 1H), 5.32 (d, J = 0.9 Hz, 1H), 4.87(s, 1H), 4.43 (q, J = 2.8 Hz, 2H), 4.17-3.94 (m, 3H), 3.03 (dd, J = 5.1,1.0 Hz, 3H), 2.76- 2.59 (m, 2H), 2.23 (d, J = 12.7 Hz, 2H), 1.98 (d, J =7.7 Hz, 6H). 321

461.63 1H NMR (300 MHz, Chloroform-d) δ 8.81- 8.67 (m, 2H), 8.42 (s,1H), 7.89 (s, 1H), 7.58 (d, J = 1.8 Hz, 1H), 7.09 (s, 1H), 6.28 (dq, J =3.0, 1.6 Hz, 1H), 4.81 (s, 1H), 4.33 (q, J = 2.8 Hz, 2H), 3.92 (t, J =5.4 Hz, 2H), 2.93 (d, J = 5.1 Hz, 3H), 2.67-2.48 (m, 2H), 2.18 (d, J =11.3 Hz, 2H), 1.98-1.73 (m, 6H). 323

435.33 324

450.35 1H NMR (300 MHz, CDCl3) δ 8.71 (d, J = 1.9 Hz, 1H), 8.63 (d, J =1.9 Hz, 1H), 7.79 (d, J = 5.9 Hz, 1H), 6.97 (d, J = 2.4 Hz, 1H), 6.91(d, J = 2.4 Hz, 1H), 6.13 (dd, J = 5.9, 2.1 Hz, 1H), 5.87 (d, J = 2.0Hz, 1H), 4.87- 4.70 (m, 1H), 4.32 (q, J = 7.1 Hz, 2H), 4.21 (d, J = 7.8Hz, 1H), 4.05-3.81 (m, 4H), 3.60-3.45 (m, 1H), 3.45-3.24 (m, 4H),2.33-2.10 (m, 2H), 2.02-1.77 (m, 6H), 1.39 (t, J = 7.1 Hz, 3H). [2] 332

465.36 333

435.33 334

451.36 335

421.37 336

446.35 340

474.48 1H NMR (300 MHz, DMSO-d6) δ 8.73 (d, J = 1.9 Hz, 1H), 8.59 (d, J= 1.9 Hz, 1H), 7.25 (m, 1H), 7.14 (d, J = 2.4 Hz, 1H), 6.89- 6.78 (m,3H), 5.47 (d, J = 7.7 Hz, 1H), 4.94 (m, 1H), 4.29 (s, 2H), 3.79 (m, 4H),3.51 (m, 1H), 3.06 (s, 3H), 2.09 (m, 2H), 1.81 (m, 6H). 341

465.57 1H NMR (300 MHz, DMSO-d6) δ 8.72 (d, J = 1.9 Hz, 1H), 8.58 (d, J= 1.9 Hz, 1H), 8.17 (s, 2H), 7.15 (d, J = 2.4 Hz, 1H), 6.83 (d, J = 2.3Hz, 1H), 6.06 (d, J = 7.8 Hz, 1H), 4.92 (m, 1H), 4.77 (s, 1H), 3.79 (m,4H), 3.50 (m, 1H), 3.30 (m, 4H), 2.02 (m, 2H), 1.80 (m, 6H), 1.42 (s,6H). 351

478.38 1H NMR (300 MHz, Chloroform-d) δ 8.70 (d, J = 1.9 Hz, 1H), 8.62(d, J = 2.0 Hz, 1H), 8.38 (d, J = 4.9 Hz, 1H), 6.93 (dd, J = 14.5, 2.4Hz, 2H), 6.68 (d, J = 4.9 Hz, 1H), 5.28 (s, 1H), 4.79 (s, 1H), 4.22-3.86(m, 5H), 3.34 (t, J = 4.8 Hz, 4H), 3.17-2.97 (m, 6H), 2.20 (s, 2H), 1.91(d, J = 5.1 Hz, 6H). 352

418.5 1H NMR (300 MHz, Chloroform-d) δ 8.91- 8.75 (m, 2H), 8.11 (d, J =5.9 Hz, 1H), 7.68 (d, J = 1.8 Hz, 1H), 6.37 (tt, J = 3.0, 1.5 Hz, 1H),6.16 (d, J = 6.0 Hz, 1H), 4.99 (s, 1H), 4.88 (d, J = 6.0 Hz, 1H), 4.43(q, J = 2.8 Hz, 2H), 4.02 (t, J = 5.4 Hz, 2H), 3.85 (s, 1H), 2.77-2.64(m, 2H), 2.51 (s, 3H), 2.23 (d, J = 13.2 Hz, 2H), 2.03-1.82 (m, 6H). 353

433.62 1H NMR (300 MHz, Chloroform-d) δ 8.67 (d, J = 1.9 Hz, 1H), 8.54(d, J = 1.9 Hz, 1H), 8.11 (d, J = 6.0 Hz, 1H), 6.85 (s, 2H), 6.16 (d, J= 6.0 Hz, 1H), 5.00 (s, 1H), 4.85 (d, J = 6.5 Hz, 3H), 3.93-3.57 (m,5H), 3.35 (q, J = 7.1 Hz, 1H), 2.62 (d, J = 8.0 Hz, 1H), 2.50 (s, 3H),2.24 (d, J = 8.9 Hz, 2H), 2.01-1.81 (m, 6H). 354

407.56 1H NMR (300 MHz, DMSO-d6) δ 8.73 (d, J = 1.9 Hz, 1H), 8.61 (d, J= 3.1 Hz, 1H), 8.58 (d, J = 2.0 Hz, 1H), 8.47 (d, J = 6.2 Hz, 1H), 7.15(d, J = 2.4 Hz, 1H), 6.99 (d, J = 7.8 Hz, 1H), 6.83 (d, J = 2.3 Hz, 1H),6.65 (m, 1H), 4.94 (m, 1H), 3.79 (m, 4H), 3.55 (m, 1H), 3.35 (m, 4H),2.03 (m, 2H), 1.78 (m, 6H). 366

475.56 369

436.5 1H NMR (300 MHz, Chloroform-d) δ 8.71 (d, J = 1.9 Hz, 1H), 8.62(d, J = 1.9 Hz, 1H), 7.71 (s, 1H), 6.94 (dd, J = 14.6, 2.5 Hz, 2H), 5.09(d, J = 7.7 Hz, 1H), 4.80 (d, J = 5.4 Hz, 1H), 4.29 (s, 2H), 4.01-3.84(m, 4H), 3.45-3.28 (m, 4H), 2.49 (s, 3H), 2.21 (q, J = 6.1 Hz, 2H), 1.95(dt, J = 10.0, 4.1 Hz, 6H). 373

421.51 1H NMR (400 MHz, CDCl3) δ 8.71 (d, J = 1.8 Hz, 1H), 8.63 (d, J =1.8 Hz, 1H), 7.70 (d, J = 5.0 Hz, 2H), 6.97 (d, J = 1.6 Hz, 1H), 6.92(d, J = 2.1 Hz, 1H), 4.81 (s, 1H), 4.65 (s, 1H), 3.99-3.85 (m, 5H),3.40-3.30 (m, 4H), 2.37 (s, 3H), 2.28- 2.16 (m, 2H), 2.01- 1.82 (m, 6H).374

441.45 375

420.57 1H NMR (300 MHz, Chloroform-d) δ 8.81- 8.67 (m, 2H), 8.01 (d, J =6.0 Hz, 1H), 7.46 (dd, J = 1.7, 0.7 Hz, 1H), 6.98 (d, J = 1.7 Hz, 1H),6.06 (d, J = 6.0 Hz, 1H), 4.80 (d, J = 38.5 Hz, 2H), 4.13- 3.97 (m, 3H),3.51 (td, J = 11.3, 3.5 Hz, 2H), 2.99-2.80 (m, 1H), 2.41 (s, 3H), 2.21-2.03 (m, 2H), 1.94- 1.70 (m, 7H). 376

455.33 1H NMR (300 MHz, Chloroform-d) δ 8.69 (d, J = 1.9 Hz, 1H), 8.62(d, J = 1.9 Hz, 1H), 8.07 (s, 1H), 6.93 (dd, J = 17.0, 2.5 Hz, 2H),4.98-4.71 (m, 2H), 4.17 (s, 1H), 4.00 (s, 3H), 3.92 (dd, J = 5.8, 3.9Hz, 4H), 3.42- 3.26 (m, 4H), 2.22 (d, J = 9.5 Hz, 2H), 1.93 (dd, J =6.1, 2.4 Hz, 6H). 377

512.36 1H NMR (300 MHz, Chloroform-d) δ 8.69 (d, J = 1.9 Hz, 1H), 8.62(d, J = 1.9 Hz, 1H), 7.95 (d, J = 1.2 Hz, 1H), 6.92 (dd, J = 16.9, 2.5Hz, 2H), 4.98 (s, 1H), 4.81-4.66 (m, 2H), 4.16 (s, 1H), 3.97- 3.87 (m,4H), 3.46 (d, J = 6.1 Hz, 2H), 3.39-3.29 (m, 4H), 2.19 (d, J = 12.5 Hz,2H), 1.91 (d, J = 5.4 Hz, 6H), 1.26 (s, 6H), 0.94-0.83 (m, 1H). 380

498.36 1H NMR (300 MHz, Chloroform-d) δ 8.69 (d, J = 2.0 Hz, 1H), 8.62(d, J = 1.9 Hz, 1H), 8.00-7.91 (m, 1H), 6.92 (dd, J = 17.2, 2.5 Hz, 2H),4.87- 4.64 (m, 2H), 4.59 (d, J = 5.7 Hz, 1H), 4.16 (ddt, J = 9.7, 6.8,3.7 Hz, 2H), 4.01- 3.85 (m, 4H), 3.75 (d, J = 11.2 Hz, 1H), 3.58 (dd, J= 10.9, 7.0 Hz, 1H), 3.48-3.23 (m, 4H), 2.36-2.07 (m, 2H), 1.91 (d, J =5.5 Hz, 6H), 1.28 (d, J = 6.8 Hz, 3H), 0.97- 0.78 (m, 1H). 382

498.32 1H NMR (300 MHz, Chloroform-d) δ 8.69 (d, J = 1.9 Hz, 1H), 8.62(d, J = 1.9 Hz, 1H), 8.00 (s, 1H), 6.92 (dd, J = 16.5, 2.5 Hz, 2H), 4.92(s, 1H), 4.77 (s, 1H), 4.64 (d, J = 8.5 Hz, 1H), 4.16 (s, 1H), 4.03-3.82(m, 4H), 3.65 (q, J = 5.8, 5.4 Hz, 2H), 3.56 (ddd, J = 5.6, 4.7, 1.0 Hz,2H), 3.48-3.21 (m, 7H), 2.19 (d, J = 10.1 Hz, 2H), 1.91 (d, J = 5.4 Hz,6H). 386

480.41 1H NMR (400 MHz, CDCl3) δ 8.70 (d, J = 1.9 Hz, 1H), 8.62 (d, J =1.9 Hz, 1H), 7.49 (s, 1H), 6.95 (d, J = 2.4 Hz, 1H), 6.89 (d, J = 2.4Hz, 1H), 5.25 (s, 1H), 4.72 (s, 1H), 4.18 (s, 1H), 3.96-3.87 (m, 4H),3.77 (s, 3H), 3.39- 3.30 (m, 4H), 3.10 (s, 6H), 2.21-2.12 (m, 2H),2.02-1.85 (m, 6H). 387

466.4 1H NMR (400 MHz, CDCl3) δ 8.70 (d, J = 1.9 Hz, 1H), 8.62 (d, J =1.9 Hz, 1H), 7.49 (s, 1H), 6.95 (d, J = 2.4 Hz, 1H), 6.89 (d, J = 2.4Hz, 1H), 5.25 (s, 1H), 4.72 (s, 1H), 4.18 (s, 1H), 3.96-3.87 (m, 4H),3.77 (s, 3H), 3.39- 3.30 (m, 4H), 3.10 (s, 6H), 2.21-2.12 (m, 2H),2.02-1.85 (m, 6H). 388

480.41 1H NMR (400 MHz, CDCl3) δ 8.69 (d, J = 1.9 Hz, 1H), 8.63 (d, J =1.9 Hz, 1H), 8.04 (s, 1H), 6.94 (d, J = 2.4 Hz, 1H), 6.89 (d, J = 2.4Hz, 1H), 5.15 (d, J = 8.4 Hz, 1H), 4.75 (s, 1H), 4.14 (s, 1H), 3.97-3.83 (m, 4H), 3.59 (s, 3H), 3.38-3.28 (m, 4H), 3.09 (s, 6H), 2.25- 2.13(m, 2H), 2.00- 1.83 (m, 6H). 393

450.58 1H NMR (300 MHz, DMSO-d6) δ 8.73 (d, J = 1.9 Hz, 1H), 8.58 (d, J= 1.9 Hz, 1H), 8.10 (d, J = 2.7 Hz, 1H), 7.97 (d, J = 8.9 Hz, 1H), 7.38(dd, J = 9.0, 2.7 Hz, 1H), 7.16 (d, J = 2.4 Hz, 1H), 6.84 (d, J = 2.3Hz, 1H), 4.96 (m, 1H), 3.79 (m, 4H), 3.66 (m, 1H), 3.3 (m, 4H),2.14-1.95 (m, 2H), 1.81 (m, 5H). 394

493.61 1H NMR (300 MHz, DMSO-d6) δ 8.72 (d, J = 1.9 Hz, 1H), 8.58 (d, J= 1.9 Hz, 1H), 7.54 (d, J = 8.6 Hz, 1H), 7.14 (d, J = 2.4 Hz, 1H), 6.83(d, J = 2.3 Hz, 1H), 6.48 (d, J = 7.7 Hz, 1H), 6.30- 6.18 (m, 2H), 4.92(m, 1H), 3.79 (m, 4H), 3.74 (s, 3H), 3.66 (s, 3H), 3.55 (m, 1H), 3.34(m, 4H), 2.03 (m, 2H), 1.78 (m, 6H). 395

441.2 1H NMR (300 MHz, Chloroform-d) δ 8.61 (d, J = 1.9 Hz, 1H), 8.53(d, J = 2.0 Hz, 1H), 7.13 (d, J = 5.3 Hz, 1H), 6.84 (dd, J = 20.0, 2.4Hz, 2H), 6.50 (d, J = 8.2 Hz, 1H), 4.72 (s, 1H), 4.31 (d, J = 7.0 Hz,1H), 3.83 (dd, J = 5.8, 3.8 Hz, 4H), 3.26 (t, J = 4.9 Hz, 4H), 2.14 (d,J = 12.3 Hz, 2H), 1.95- 1.68 (m, 6H). 396

455.2 1H NMR (300 MHz, Chloroform-d) δ 8.61 (d, J = 1.9 Hz, 1H), 8.54(d, J = 1.9 Hz, 1H), 7.72 (d, J = 2.9 Hz, 1H), 6.87 (d, J = 2.5 Hz, 1H),6.81 (d, J = 2.5 Hz, 1H), 5.03 (d, J = 7.8 Hz, 1H), 4.71 (s, 1H), 4.16(s, 2H), 3.83 (q, J = 3.9, 3.1 Hz, 7H), 3.32-3.21 (m, 4H), 2.13 (d, J =11.2 Hz, 2H), 1.83 (t, J = 6.5 Hz, 6H). 397

422.5 1H NMR (300 MHz, Chloroform-d) δ 8.70 (d, J = 1.9 Hz, 1H), 8.63(d, J = 1.9 Hz, 1H), 7.89 (d, J = 5.8 Hz, 1H), 7.02-6.88 (m, 2H), 5.79(d, J = 5.7 Hz, 1H), 4.80 (s, 1H), 4.61 (s, 2H), 4.08- 3.88 (m, 5H),3.35 (dd, J = 5.6, 4.1 Hz, 4H), 2.27-2.09 (m, 2H), 1.91 (d, J = 5.0 Hz,6H). 399

435.35 1H NMR (300 MHz, CDCl3) δ 8.69 (d, J = 1.9 Hz, 1H), 8.61 (d, J =1.9 Hz, 1H), 8.08 (d, J = 6.3 Hz, 1H), 6.96 (d, J = 2.5 Hz, 1H), 6.92(d, J = 2.4 Hz, 1H), 6.23 (d, J = 6.3 Hz, 1H), 4.87 (s, 1H), 4.69 (s,1H), 4.00- 3.83 (m, 4H), 3.45- 3.24 (m, 4H), 2.98 (s, 3H), 2.50 (s, 3H),2.45- 2.30 (m, 2H), 1.92- 1.64 (m, 6H). 401

471.32 1H NMR (300 MHz, CDCl3) δ 8.69 (d, J = 1.9 Hz, 1H), 8.62 (d, J =1.9 Hz, 1H), 7.51 (s, 1H), 6.95 (d, J = 2.4 Hz, 1H), 6.89 (d, J = 2.4Hz, 1H), 5.56 (d, J = 8.0 Hz, 1H), 4.75 (s, 1H), 4.25 (s, 1H), 3.96-3.89 (m, 4H), 3.88 (s, 3H), 3.47-3.23 (m, 4H), 2.29-2.06 (m, 2H),2.06-1.78 (m, 6H). 402

471.36 1H NMR (300 MHz, CDCl3) δ 8.70 (d, J = 1.9 Hz, 1H), 8.62 (d, J =1.9 Hz, 1H), 8.14 (s, 1H), 6.96 (d, J = 2.4 Hz, 1H), 6.90 (d, J = 2.5Hz, 1H), 5.52 (d, J = 8.6 Hz, 1H), 4.79 (s, 1H), 4.18 (s, 1H), 3.96-3.89 (m, 4H), 3.89 (s, 3H), 3.41-3.27 (m, 4H), 2.31-2.14 (m, 2H),2.03-1.83 (m, 6H). 404

480.56 1H NMR (300 MHz, DMSO-d6) δ 8.72 (d, J = 1.9 Hz, 1H), 8.57 (d, J= 1.9 Hz, 1H), 7.69 (s, 1H), 7.15 (d, J = 2.5 Hz, 1H), 7.01 (s, 1H),6.84 (d, J = 2.3 Hz, 1H), 4.94 (m, 1H), 3.75 (m, 8H), 3.34 (m, 4H), 2.05(m, 2H), 1.86-1.70 (m, 6H). 405

480.56 1H NMR (300 MHz, DMSO-d6) δ 8.72 (d, J = 1.9 Hz, 1H), 8.57 (d, J= 1.9 Hz, 1H), 8.01 (m, 1H), 7.14 (s, 1H), 7.01-6.90 (m, 1H), 6.82 (m,2H), 6.71 (m, 1H), 5.70 (d, J = 7.9 Hz, 1H), 4.91 (m, 1H), 3.79 (m, 4H),3.34 (m, 5H), 2.74 (d, J = 4.6 Hz, 3H), 2.02 (m ,2H), 1.76 (m, 6H). 406

463.68 1H NMR (300 MHz, DMSO-d6) δ 8.72 (d, J = 1.9 Hz, 1H), 8.58 (d, J= 1.9 Hz, 1H), 8.27 (d, J = 5.2 Hz, 1H), 7.46 (dd, J = 8.4, 7.1 Hz, 1H),7.15 (d, J = 2.4 Hz, 1H), 7.10 (dd, J = 7.1, 0.9 Hz, 1H), 6.83 (d, J =2.3 Hz, 1H), 6.75 (d, J = 7.8 Hz, 1H), 6.68 (dd, J = 8.4, 0.9 Hz, 1H),4.91 (m, 1H), 4.14 (m, 1H), 3.80 (m, 4H), 3.35 (m, 4H), 2.82 (d, J = 4.9Hz, 3H), 2.03 (m, 2H), 1.93-1.73 (m, 6H). 407

536.49 1H NMR (300 MHz, Chloroform-d) rotomers, δ 9.07 (m, 1H), 8.89 (m,1H), 8.75 (m, 1H), 7.47 (m, 1H), 6.96 (s, 1H), 4.95 (m, 1H), 3.97 (m,4H), 3.82 (m, 4H), 3.38 (s, 3H), 2.24 (m, 8H), 1.60 (s, 9H). 408

435.6 1H NMR (300 MHz, DMSO-d6) δ 8.72 (d, J = 1.9 Hz, 1H), 8.57 (d, J =1.9 Hz, 1H), 7.13 (d, J = 2.4 Hz, 1H), 6.94 (t, J = 8.0 Hz, 1H), 6.83(d, J = 2.3 Hz, 1H), 6.26-6.14 (m, 2H), 6.10-6.03 (m, 1H), 5.57 (d, J =7.8 Hz, 1H), 4.90 (m, 1H), 3.79 (m, 4H), 3.66 (s, 3H), 3.33 (m, 4H),2.03 (m, 2H), 1.77 (m, 6H). 409

441.29 1H NMR (300 MHz, CDCl3) δ 8.70 (d, J = 1.9 Hz, 1H), 8.63 (d, J =1.9 Hz, 1H), 8.47 (s, 1H), 8.18 (s, 1H, 6.96 (d, J = 2.4 Hz, 1H), 6.91(d, J = 2.4 Hz, 1H), 5.47 (d, J = 7.6 Hz, 1H), 4.80 (s, 1H), 4.31-4.19(m, 1H), 4.02-3.79 (m, 4H), 3.48-3.24 (m, 4H), 2.32-2.17 (m, 2H),2.09-1.79 (m, 6H). 417

475.36 1H NMR (300 MHz, Chloroform-d) δ 8.81- 8.55 (m, 3H), 6.94 (dd, J= 17.2, 2.5 Hz, 2H), 6.65 (d, J = 1.1 Hz, 1H), 4.84 (td, J = 5.1, 2.4Hz, 1H), 4.01- 3.86 (m, 4H), 3.42- 3.29 (m, 4H), 2.35- 2.11 (m, 2H),2.05- 1.82 (m, 6H). 418

475.4 1H NMR (300 MHz, Chloroform-d) δ 8.71 (d, J = 1.9 Hz, 1H), 8.63(d, J = 1.9 Hz, 1H), 8.49 (d, J = 4.9 Hz, 1H), 7.04-6.89 (m, 2H), 6.81(d, J = 4.9 Hz, 1H), 5.54 (d, J = 7.9 Hz, 1H), 4.80 (d, J = 5.2 Hz, 1H),4.17- 3.98 (m, 1H), 3.98- 3.87 (m, 4H), 3.42- 3.31 (m, 4H), 2.33- 2.12(m, 2H), 2.06- 1.72 (m, 6H). 419

450.38 1H NMR (300 MHz, Chloroform-d) rotomers δ 8.82 (d, J = 2.9 Hz,1H), 8.74 (m, 1H), 8.00 (m, 1H), 7.55-7.47 (m, 1H), 7.25 (m, 1H), 7.14(m, 1H), 7.05 (m, 1H), 3.97 (m, 5H), 3.47 (m, 5H), 2.30 (s, 2H), 2.0 (m,6H).. 420

467.48 1H NMR (300 MHz, DMSO-d6) δ 8.73 (d, J = 1.9 Hz, 1H), 8.58 (d, J= 2.0 Hz, 1H), 7.27- 6.96 (m, 4H), 6.83 (d, J = 2.3 Hz, 1H), 4.92 (m,1H), 3.85-3.74 (m, 4H), 3.45 (m, 1H), 3.34 (m, 4H), 2.05 (m, 2H), 1.76(m, 6H). 421

523.58 1H NMR (300 MHz, DMSO-d6) δ 8.93 (d, J = 1.8 Hz, 1H), 8.80 (d, J= 1.8 Hz, 1H), 7.20 (s, 1H), 7.13-6.85 (m, 4H), 4.92 (m, 1H), 3.86-3.79(m, 4H), 3.32 (m, 5H), 2.09 (m, 2H), 1.90-1.73 (m, 6H), 1.55 (s, 9H).422

432.5 1H NMR (300 MHz, Chloroform-d) δ 8.72- 8.52 (m, 3H), 8.38 (s, 1H),6.87 (dd, J = 15.7, 2.5 Hz, 2H), 5.50 (d, J = 7.9 Hz, 1H), 4.79 (p, J =3.5 Hz, 1H), 4.20 (dp, J = 14.0, 5.2, 4.7 Hz, 1H), 3.90-3.76 (m, 4H),3.32-3.22 (m, 4H), 2.29-2.07 (m, 2H), 1.99-1.71 (m, 6H). 424

437.3 1H NMR (300 MHz, Chloroform-d) δ 8.62 (d, J = 1.9 Hz, 1H), 8.52(d, J = 1.9 Hz, 1H), 8.46-8.35 (m, 1H), 6.84 (dd, J = 16.8, 2.5 Hz, 2H),6.28- 6.20 (m, 1H), 5.08 (s, 1H), 4.72 (d, J = 5.2 Hz, 1H), 4.58-4.43(m, 2H), 3.93-3.77 (m, 4H), 3.34-3.19 (m, 4H), 2.14 (dd, J = 9.6, 5.4Hz, 2H), 1.93- 1.73 (m, 6H). 427

474.33 1H NMR (300 MHz, Choroform-d) δ 8.94- 8.63 (m, 2H), 8.07 (d, J =2.8 Hz, 1H), 7.45 (d, J = 8.7 Hz, 1H), 7.14-6.87 (m, 3H), 4.84 (m, 1H),3.99- 3.87 (m, 4H), 3.62- 3.48 (m, 1H), 3.35 (dt, J = 42.8, 4.8 Hz, 4H),2.32-2.14 (m, 2H), 1.91 (d, J = 17.8 Hz, 6H). 428

475.41 1H NMR (300 MHz, Chloroform-d) δ 8.81- 8.64 (m, 2H), 8.20 (s,2H), 7.08 (d, J = 2.4 Hz, 1H), 6.94 (d, J = 2.4 Hz, 1H), 4.90- 4.82 (m,1H), 4.42 (m, 1H), 3.98-3.88 (m, 4H), 3.57 (m, 1H), 3.45-3.26 (m, 4H),2.33-2.18 (m, 2H), 2.04-1.82 (m, 6H). 429

474.37 1H NMR (300 MHz, Chloroform-d) δ 8.70 (d, J = 2.0 Hz, 1H), 8.63(d, J = 2.0 Hz, 1H), 8.42 (m, 1H), 8.16-8.08 (m, 1H), 7.20 (s, 1H),7.04- 6.98 (m, 1H), 6.94 (d, J = 2.4 Hz, 1H), 4.84 (m, 1H), 4.01-3.87(m, 4H), 3.54 (m, 1H), 3.42-3.26 (m, 4H), 2.33-2.19 (m, 2H), 2.08-1.80(m, 6H). 430

533.47 1H NMR (300 MHz, Chloroform-d) δ 8.69 (d, J = 1.9 Hz, 1H), 8.62(d, J = 1.9 Hz, 1H), 8.15 (s, 2H), 7.03- 6.85 (m, 2H), 5.09 (d, J = 8.1Hz, 1H), 4.78 (s, 1H), 3.92 (dd, J = 5.9, 3.7 Hz, 5H), 3.40-3.24 (m,4H), 2.71-2.33 (m, 8H), 2.30 (s, 3H), 2.20 (s, 2H), 1.91 (d, J = 5.0 Hz,6H), 1.26 (d, J = 3.1 Hz, 4H). 436

449.44 1H NMR (400 MHz, CDCl3) δ 8.70 (d, J = 1.9 Hz, 1H), 8.61 (d, J =1.9 Hz, 1H), 6.95 (d, J = 2.4 Hz, 1H), 6.90 (d, J = 2.4 Hz, 1H),4.88-4.70 (m, 2H), 4.32 (s, 1H), 3.98- 3.85 (m, 4H), 3.41- 3.27 (m, 4H),2.53 (s, 3H), 2.40 (s, 3H), 2.26- 2.14 (m, 2H), 1.99 (s, 3H), 1.94-1.88(m, 6H). 438

437.3 1H NMR (300 MHz, Chloroform-d) δ 8.72 (d, J = 2.0 Hz, 1H), 8.64(d, J = 1.9 Hz, 1H), 8.28 (s, 1H), 7.72 (s, 1H), 6.94 (dd, J = 16.4, 2.5Hz, 2H), 5.45 (d, J = 8.2 Hz, 1H), 4.76 (d, J = 6.6 Hz, 1H), 4.26 (s,1H), 4.01- 3.85 (m, 7H), 3.44- 3.28 (m, 4H), 2.30- 2.11 (m, 2H), 2.08-1.82 (m, 6H). 439

435.44 1H NMR (400 MHz, CDCl3) δ 8.71 (d, J = 1.8 Hz, 1H), 8.63 (d, J =1.8 Hz, 1H), 8.14 (d, J = 5.9 Hz, 1H), 6.98 (d, J = 2.4 Hz, 1H), 6.17(d, J = 6.0 Hz, 1H), 5.05 (s, 1H), 4.01- 3.70 (m, 5H), 3.42- 3.25 (m,4H), 2.77 (q, J = 7.6 Hz, 2H), 2.32- 2.16 (m, 2H), 2.04- 1.80 (m, 6H),1.32 (t, J = 7.6 Hz, 3H). 440

436.22 1H NMR (300 MHz, Chloroform-d) δ 8.70 (d, J = 1.9 Hz, 1H), 8.60(d, J = 1.9 Hz, 1H), 7.81 (d, J = 6.3 Hz, 1H), 6.96 (d, J = 2.4 Hz, 1H),6.90 (d, J = 2.6 Hz, 1H), 6.24 (d, J = 6.2 Hz, 1H), 5.92 (s, 1H), 4.80(m, 1H), 3.98 (s, 3H), 3.95- 3.87 (m, 4H), 3.54 (m, 1H), 3.38-3.28 (m,4H), 2.23 (m, 2H), 2.00-1.81 (m, 6H). 441

434.35 1H NMR (300 MHz, Chloroform-d) rotomers, δ 8.80 (d, 1H), 8.73 (d,1H), 7.73 (s, 1H), 7.16-6.99 (m, 3H), 4.91 (m, 1H), 3.95 (m, 4H), 3.55(m, 1H), 3.37 (m, 4H), 2.73 (s, 3H), 2.41 (s, 3H), 2.29 (m, 2H), 2.13(m, 6H). 442

420.4 1H NMR (300 MHz, Chloroform-d) δ 8.70 (d, J = 1.9 Hz, 1H), 8.62(d, J = 1.9 Hz, 1H), 7.89 (dd, J = 5.0, 1.4 Hz, 1H), 7.19- 7.10 (m, 1H),7.03- 6.95 (m, 2H), 6.92 (d, J = 2.6 Hz, 1H), 4.80 (m, 1H), 3.96-3.87(m, 4H), 3.79 (m, 1H), 3.51 (m, 1H), 3.38- 3.29 (m, 4H), 2.51 (m, 3H),2.25 (m, 2H), 2.00-1.87 (m, 6H). 443

420.36 1H NMR (300 MHz, Chloroform-d) δ 8.69 (d, J = 1.9 Hz, 1H), 8.61(d, J = 1.9 Hz, 1H), 8.01 (s, 1H), 7.89 (d, J = 4.8 Hz, 1H), 7.03 (d, J= 4.8 Hz, 1H), 6.96 (d, J = 2.4 Hz, 1H), 6.91 (d, J = 2.5 Hz, 1H), 4.78(m, 1H), 3.98-3.86 (m, 4H), 3.61 (m, 2H), 3.39-3.28 (m, 4H), 2.30-2.15(m, 5H), 1.93 (m, 6H). 444

420.28 1H NMR (300 MHz, Chloroform-d) δ 8.69 (d, J = 1.9 Hz, 1H), 8.60(d, J = 1.9 Hz, 1H), 8.03 (dd, J = 2.7, 0.9 Hz, 1H), 7.12- 7.00 (m, 2H),6.95 (d, J = 2.5 Hz, 1H), 6.90 (d, J = 2.5 Hz, 1H), 4.79 (m, 1H), 4.08-3.87 (m, 5H), 3.49 (m, 1H), 3.38-3.28 (m, 4H), 2.53 (s, 3H), 2.21 (m,2H), 1.84 (d, J = 6.8 Hz, 6H). 445

436.43 1H NMR (300 MHz, Chloroform-d) δ 8.65 (m, 1H), 8.40 (m, 1H), 7.50(dd, J = 4.5, 2.2 Hz, 1H), 7.03 (s, 1H), 6.92 (s, 1H), 6.80 (m, 2H),4.76 (m, 1H), 4.08 (s, 3H), 3.93 (m, 5H), 3.49 (m, 1H), 3.42-3.30 (m,4H), 2.20 (m, 2H), 1.92 (m, 6H). 446

436.17 1H NMR (300 MHz, Chloroform-d) δ 8.69 (d, J = 1.9 Hz, 1H), 8.60(d, J = 1.9 Hz, 1H), 7.71 (d, J = 2.4 Hz, 1H), 7.65 (d, J = 2.4 Hz, 1H),6.96 (d, J = 2.4 Hz, 1H), 6.90 (d, J = 2.4 Hz, 1H), 6.55 (t, J = 2.4 Hz,1H), 4.79 (m, 1H), 4.13 (m, 1H), 3.92 (m, 4H), 3.85 (s, 3H), 3.48 (m,1H), 3.38-3.29 (m, 4H), 2.22 (m, 2H), 1.98-1.88 (m, 6H). 447

436.34 1H NMR (300 MHz, Chloroform-d) δ 8.69 (d, J = 1.9 Hz, 1H), 8.60(d, J = 2.0 Hz, 1H), 7.70 (s, 1H), 7.15 (m, 1H), 6.96 (d, J = 2.4 Hz,1H), 6.92 (m, 1H), 6.64 (d, J = 8.8 Hz, 1H), 4.80 (m, 1H), 3.90 (m, 7H),3.45- 3.26 (m, 5H), 2.24- 2.12 (m, 2H), 1.98- 1.81 (m, 6H). 448

421.39 1H NMR (300 MHz, Chloroform-d) δ 8.78 (dd, J = 4.0, 2.0 Hz, 1H),8.66 (d, J = 2.1 Hz, 1H), 8.55 (s, 1H), 8.06 (s, 1H), 7.02 (d, J = 2.5Hz, 1H), 6.96 (d, J = 2.5 Hz, 1H), 4.88 (m, 1H), 3.93 (m, 4H), 3.55 (m,1H), 3.40 (m, 4H), 3.29 (m, 1H), 2.58 (s, 3H), 2.28 (m, 2H), 2.07 (m,6H). 449

435.35 1H NMR (300 MHz, Chloroform-d) rotomers, δ 8.85 (d, 1H), 8.67 d,1H), 7.92 (d, 1H), 7.07-6.96 (m, 2H), 4.91 (m, 1H), 4.01-3.88 (m, 4H),3.53-3.24 (m, 5H), 2.87 (s, 3H), 2.64 (s, 3H), 2.04-1.76 (m, 6H). 450

421.36 1H NMR (300 MHz, Chloroform-d) δ 8.69 (d, J = 1.9 Hz, 1H), 8.61(d, J = 1.9 Hz, 1H), 7.21 (d, J = 9.2 Hz, 1H), 6.99-6.87 (m, 3H), 4.87(m, 1H), 398-3.82 (m, 5H), 3.39-3.29 (m, 4H), 2.51 (s, 3H), 2.30- 2.22(m, 2H), 2.11- 1.84 (m, 6H). 451

421.56 1H NMR (300 MHz, Chloroform-d) rotomers, δ 8.92 (d, J = 1.8 Hz,1H), 8.77 (d, J = 1.8 Hz, 1H), 8.30 (m, 2H), 7.02-6.90 (m, 2H), 4.86 (m,1H), 3.94 (m, 4H), 3.52- 3.26 (m, 5H), 2.78 (s, 3H), 2.26 (d, J = 11.8Hz, 2H), 1.90 (m, 6H). 453

450.43 1H NMR (300 MHz, CDCl3) δ 8.69 (s, 1H), 8.62 (s, 1H), 7.89 (d, J= 5.9 Hz, 1H), 6.94 (s, 1H), 6.89 (s, 1H), 5.64 (d, J = 6.0 Hz, 1H),4.87-4.66 (m, 2H), 3.98-3.83 (m, 4H), 3.44-3.24 (m, 4H), 3.13 (s, 6H),2.26- 2.10 (m, 2H), 2.04- 1.75 (m, 6H). 454

436.34 1H NMR (300 MHz, CDCl3) δ 8.61 (d, J = 1.8 Hz, 1H), 8.52 (d, J =1.9 Hz, 1H), 7.69 (s, 1H), 6.86 (d, J = 2.4 Hz, 1H), 6.80 (d, J = 2.2Hz, 1H), 5.62 (d, J = 6.0 Hz, 1H), 4.83 (s, 1H), 4.69 (s, 1H), 3.91-3.74 (m, 4H), 3.36- 3.10 (m, 4H), 2.86 (d, J = 5.0 Hz, 3H), 2.22- 2.03(m, 2H), 1.91- 1.72 (m, 6H). 455

447.41 1H NMR (300 MHz, CDCl3) δ 8.69 (d, J = 1.9 Hz, 1H), 8.61 (d, J =1.9 Hz, 1H), 8.05 (d, J = 5.2 Hz, 1H), 6.94 (d, J = 2.5 Hz, 1H), 6.89(d, J = 2.5 Hz, 1H), 6.39 (d, J = 5.2 Hz, 1H), 5.13 (bd, 1H), 4.76 (s,1H), 4.10- 3.96 (m, 1H), 3.95- 3.83 (m, 4H), 3.40- 3.24 (m, 4H), 2.26-2.09 (m, 2H), 1.97- 1.76 (m, 7H), 1.10- 1.00 (m, 2H), 1.00- 0.91 (m,2H). 456

465.29 1H NMR (300 MHz, CDCl3) δ 8.72 (d, J = 1.9 Hz, 1H), 8.64 (d, J =1.9 Hz, 1H), 8.23 (d, J = 1.8 Hz, 1H), 6.97 (d, J = 2.5 Hz, 1H), 6.92(d, J = 2.4 Hz, 1H), 5.04 (d, J = 7.6 Hz, 1H), 4.81 (s, 1H), 4.21 (s,1H), 4.02- 3.82 (m, 4H), 3.44- 3.26 (m, 4H), 2.33- 2.10 (m, 3H), 2.06-1.82 (m, 6H), 1.19- 1.11 (m, 2H), 1.06- 0.97 (m, 2H). 457

437.38 1H NMR (300 MHz, CDCl3) δ 8.69 (d, J = 1.9 Hz, 1H), 8.62 (d, J =1.9 Hz, 1H), 8.21 (d, J = 51H NMR (300 MHz, CDCl3) δ 8.69 (d, J = 1.9Hz, 1H), 8.62 (d, J = 1.9 Hz, 1H), 8.21 (d, J = 5.1 Hz, 1H), 6.95 (d, J= 2.5 Hz, 1H), 6.90 (d, J = 2.5 Hz, 1H), 6.45 (d, J = 5.1 Hz, 1H), 5.33(d, J = 7.6 Hz, 1H), 4.85-4.74 (m, 1H), 4.56 (s, 2H), 4.12- 4.00 (m,1H), 3.95- 3.86 (m, 4H), 3.66 (s, 1H), 3.39-3.27 (m, 4H), 2.18 (dd, J =14.1, 8.4 Hz, 2H), 2.01- 1.83 (m, 6H). 463

453.4 1H NMR (400 MHz, CDCl3) δ 8.69 (d, J = 1.9 Hz, 1H), 8.62 (d, J =1.9 Hz, 1H), 6.95 (d, J = 2.4 Hz, 1H), 6.90 (d, J = 2.4 Hz, 1H), 4.95(d, J = 7.1 Hz, 1H), 4.78 (s, 1H), 4.23 (d, J = 3.8 Hz, 1H), 3.99-3.82(m, 4H), 3.42-3.25 (m, 4H), 2.45 (d, J = 0.6 Hz, 3H), 2.30 (d, J = 2.8Hz, 3H), 2.26-2.14 (m, 2H), 1.99-1.86 (m, 6H). 465

420.19 1H NMR (300 MHz, Chloroform-d) δ 8.70 (d, J = 1.9 Hz, 1H), 8.61(d, J = 1.9 Hz, 1H), 7.86 (d, J = 2.7 Hz, 1H), 7.78 (s, 1H), 6.96 (d, J= 2.4 Hz, 1H), 6.90 (d, J = 2.5 Hz, 1H), 6.78 (t, J = 2.1 Hz, 1H), 4.78(m, 1H), 4.00-3.86 (m, 4H), 3.50 (s, 1H), 3.38- 3.28 (m, 4H), 2.28 (s,3H), 2.25-2.14 (m, 2H), 1.95-1.85 (m, 6H). 467

435.35 1H NMR (400 MHz, CDCl3) δ 8.68 (d, J = 1.9 Hz, 1H), 8.61 (d, J =1.9 Hz, 1H), 7.95 (s, 1H), 6.94 (d, J = 2.5 Hz, 1H), 6.90 (d, J = 2.5Hz, 1H), 5.00 (d, J = 7.9 Hz, 1H), 4.83- 4.74 (m, 1H), 4.07- 3.96 (m,1H), 3.96- 3.87 (m, 4H), 3.38- 3.28 (m, 4H), 2.29 (s, 3H), 2.22-2.12 (m,2H), 2.07 (s, 3H), 1.93- 1.85 (m, 6H). 469

454.3 1H NMR (400 MHz, CDCl3) δ 8.69 (d, J = 1.9 Hz, 1H), 8.62 (d, J =1.9 Hz, 1H), 8.03 (s, 1H), 6.95 (d, J = 2.4 Hz, 1H), 6.89 (d, J = 2.4Hz, 1H), 4.77 (s, 1H), 4.67 (s, 1H), 4.60 (s, 1H), 4.16 (s, 1H),4.00-3.82 (m, 4H), 3.41-3.25 (m, 4H), 3.04 (d, J = 5.0 Hz, 3H),2.28-2.11 (m, 2H), 1.99-1.84 (m, 6H). 471

421.35 1H NMR (300 MHz, Chloroform-d) δ 8.62 (d, J = 1.9 Hz, 1H), 8.54(d, J = 2.0 Hz, 1H), 8.42 (s, 1H), 7.91 (d, J = 13.5 Hz, 1H), 6.93-6.78(m, 2H), 4.71 (s, 1H), 4.57 (d, J = 7.9 Hz, 1H), 4.22 (s, 1H), 3.91-3.78(m, 4H), 3.33-3.19 (m, 4H), 2.16 (d, J = 6.6 Hz, 2H), 1.96 (s, 3H),1.91-1.77 (m, 6H). 472

534.2 1H NMR (300 MHz, Chloroform-d) δ 8.62 (d, J = 1.9 Hz, 1H), 8.54(d, J = 1.9 Hz, 1H), 8.10 (d, J = 0.8 Hz, 1H), 6.92-6.77 (m, 2H), 5.36(dd, J = 7.9, 0.9 Hz, 1H), 4.71 (q, J = 5.2, 4.2 Hz, 2H), 3.97-3.69 (m,7H), 3.58-3.37 (m, 4H), 3.30-3.03 (m, 6H), 2.22-2.04 (m, 2H), 1.93-1.73(m, 6H), 1.52 (dtd, J = 12.8, 8.9, 3.8 Hz, 2H), 1.28-1.16 (m, 3H). 474

523.31 1H NMR (400 MHz, CDCl3) δ 8.69 (d, J = 1.9 Hz, 1H), 8.61 (d, J =1.9 Hz, 1H), 7.99 (d, J = 1.5 Hz, 1H), 6.94 (d, J = 2.4 Hz, 1H), 6.89(d, J = 2.4 Hz, 1H), 4.82 (d, J = 5.5 Hz, 1H), 4.77 (s, 1H), 4.15 (s,1H), 3.96- 3.85 (m, 4H), 3.76- 3.65 (m, 4H), 3.38- 3.26 (m, 4H), 2.59-2.45 (m, 4H), 2.34 (s, 3H), 2.27-2.13 (m, 2H), 1.96-1.85 (m, 6H). 476

439.4 1H NMR (400 MHz, CDCl3) δ 8.70 (d, J = 1.9 Hz, 1H), 8.63 (d, J =1.9 Hz, 1H), 8.03 (d, J = 1.7 Hz, 1H), 6.97 (d, J = 2.5 Hz, 1H), 6.92(d, J = 2.5 Hz, 1H), 5.18-5.04 (m, 1H), 4.80 (s, 1H), 3.99- 3.90 (m,5H), 3.40- 3.32 (m, 4H), 2.36 (d, J = 2.4 Hz, 3H), 2.23- 2.15 (m, 2H),1.95- 1.85 (m, 6H). 477

439.36 1H NMR (400 MHz, CDCl3) δ 8.70 (d, J = 1.9 Hz, 1H), 8.62 (d, J =1.9 Hz, 1H), 8.26 (d, J = 1.7 Hz, 1H), 6.95 (d, J = 2.4 Hz, 1H), 6.90(d, J = 2.4 Hz, 1H), 5.07 (d, J = 7.2 Hz, 1H), 4.80 (s, 1H), 4.30-4.13(m, 1H), 3.98-3.83 (m, 4H), 3.38-3.26 (m, 4H), 2.36 (d, J = 2.8 Hz, 3H),2.29-2.16 (m, 2H), 1.97-1.85 (m, 6H). 478

424 1H NMR (400 MHz, CDCl3) δ 8.64 (t, J = 9.6 Hz, 1H), 8.53 (d, J = 1.9Hz, 1H), 8.02 (d, J = 5.9 Hz, 1H), 6.89 (d, J = 2.4 Hz, 1H), 6.83 (d, J= 2.4 Hz, 1H), 6.07 (d, J = 6.0 Hz, 1H), 4.91 (s, 1H), 4.73 (s, 1H),3.83 (dd, J = 17.8, 12.9 Hz, 5H), 3.25 (dd, J = 17.8, 13.0 Hz, 4H), 2.14(dd, J = 8.9, 5.0 Hz, 2H), 1.91-1.76 (m, 6H). 481

451.41 1H NMR (300 MHz, Chloroform-d) δ 8.69 (d, J = 1.9 Hz, 1H), 8.62(d, J = 1.9 Hz, 1H), 8.18 (s, 2H), 6.99- 6.87 (m, 2H), 5.16 (d, J = 8.1Hz, 1H), 4.78 (s, 1H), 4.03- 3.87 (m, 5H), 3.80 (t, J = 6.5 Hz, 2H),3.39- 3.29 (m, 4H), 2.67 (t, J = 6.5 Hz, 2H), 2.26- 2.14 (m, 2H), 1.90(t, J = 6.5 Hz, 6H). 482

454.35 1H NMR (400 MHz, CDCl3) δ 8.68 (d, J = 1.9 Hz, 1H), 8.61 (d, J =1.9 Hz, 1H), 7.65 (d, J = 3.6 Hz, 1H), 6.94 (d, J = 2.4 Hz, 1H), 6.90(d, J = 2.4 Hz, 1H), 5.14 (s, 1H), 4.90 (s, 1H), 4.76 (s, 1H), 4.01-3.87(m, 5H), 3.39-3.31 (m, 4H), 3.01 (d, J = 5.0 Hz, 3H), 2.23-2.12 (m, 2H),1.99-1.81 (m, 6H). 485

441.29 1H NMR (400 MHz, CDCl3) δ 8.68 (d, J = 1.9 Hz, 1H), 8.62 (d, J =1.9 Hz, 1H), 8.20 (s, 2H), 6.96 (d, J = 2.3 Hz, 1H), 6.90 (d, J = 2.4Hz, 1H), 5.27 (d, J = 7.4 Hz, 1H), 4.79 (s, 1H), 4.01-3.84 (m, 5H),3.40-3.25 (m, 4H), 2.25-2.12 (m, 2H), 1.97-1.82 (m, 6H). 486

407 1H NMR (400 MHz, CDCl3) δ 8.62 (d, J = 1.9 Hz, 1H), 8.54 (d, J = 1.9Hz, 1H), 8.49 (s, 1H), 8.08 (d, J = 6.0 Hz, 1H), 6.89 (d, J = 2.5 Hz,1H), 6.83 (d, J = 2.5 Hz, 1H), 6.24 (dd, J = 6.0, 1.1 Hz, 1H), 4.88 (s,1H), 4.78- 4.67 (m, 1H), 3.87- 3.82 (m, 4H), 3.25 (dd, J = 13.6, 8.8 Hz,4H), 2.15 (dd, J = 8.8, 5.1 Hz, 2H), 1.90-1.78 (m, 6H). 489

450.34 1H NMR (400 MHz, CDCl3) δ 8.68 (d, J = 1.9 Hz, 1H), 8.61 (d, J =1.9 Hz, 1H), 7.85 (d, J = 6.1 Hz, 1H), 6.94 (d, J = 2.4 Hz, 1H), 6.90(d, J = 2.4 Hz, 1H), 5.81 (d, J = 6.1 Hz, 1H), 4.92 (s, 1H), 4.75 (s,1H), 4.02 (s, 1H), 3.97-3.86 (m, 4H), 3.32 (dd, J = 17.6, 12.8 Hz, 4H),3.04 (s, 6H), 2.18 (dd, J = 24.2, 17.3 Hz, 2H). 490

435.35 1H NMR (400 MHz, CDCl3) δ 8.70 (d, J = 1.9 Hz, 1H), 8.62 (d, J =1.9 Hz, 1H), 7.87 (d, J = 0.7 Hz 1H), 6.95 (d, J = 2.4 Hz, 1H), 6.90 (d,J = 2.4 Hz, 1H), 4.78 (s, 1H), 4.67 (s, 1H), 4.41-4.28 (m, 1H),3.96-3.88 (m, 4H), 3.38-3.30 (m, 4H), 2.52 (s, 3H), 2.28- 2.16 (m, 2H),2.00 (s, 3H), 1.98-1.90 (m, 6H). 491

435.35 1H NMR (400 MHz, CDCl3) δ 8.70 (d, J = 1.7 Hz, 1H), 8.61 (d, J =1.7 Hz, 1H), 8.42 (s, 1H), 6.95 (s, 1H), 6.90 (s, 1H), 4.79 (s, 2H),4.28 (s, 1H), 3.98- 3.86 (m, 4H), 3.40- 3.28 (m, 4H), 2.42 (s, 3H),2.30-2.16 (m, 2H), 2.02 (s, 3H), 1.99- 1.89 (m, 6H). 492

506.2 1H NMR (300 MHz, Chloroform-d) δ 8.71 (d, J = 1.9 Hz, 1H), 8.63(d, J = 1.9 Hz, 1H), 8.20 (d, J = 0.8 Hz, 1H), 6.97 (d, J = 2.4 Hz, 1H),6.92 (d, J = 2.5 Hz, 1H), 5.47 (d, J = 1.0 Hz, 1H), 4.76 (d, J = 23.9Hz, 2H), 4.12-3.75 (m, 7H), 3.41-3.28 (m, 4H), 3.20 (ddd, J = 13.2, 9.6,3.2 Hz, 2H), 2.20 (d, J = 6.9 Hz, 2H), 1.91 (d, J= 5.2 Hz, 6H). 494

439.23 1H NMR (300 MHz, Chloroform-d) δ 8.62 (d, J = 1.9 Hz, 1H), 8.54(d, J = 1.9 Hz, 1H), 7.82 (d, J = 3.4 Hz, 1H), 6.88 (d, J = 2.5 Hz, 1H),6.82 (d, J = 2.5 Hz, 1H), 4.96 (d, J = 8.0 Hz, 1H), 4.72 (d, J = 3.1 Hz,1H), 4.18 (s, 1H), 3.90- 3.78 (m, 4H), 3.35- 3.19 (m, 4H), 2.41 (d, J =0.9 Hz, 3H), 2.13- (q, J = 6.4 Hz, 2H), 1.84 (t, J = 6.3 Hz, 6H). 499

535.24 1H NMR (300 MHz, Chloroform-d) δ 8.70 (d, J = 1.9 Hz, 1H), 8.61(d, J = 1.9 Hz, 1H), 8.19 (d, J = 0.8 Hz, 1H), 6.93 (dd, J = 16.4, 2.5Hz, 2H), 5.42 (d, J = 1.0 Hz, 1H), 4.87-4.67 (m, 2H), 4.01-3.77 (m, 5H),3.61 (dt, J = 27.3, 5.1 Hz, 6H), 3.41-3.26 (m, 4H), 2.73 (t, J = 5.3 Hz,1H), 2.59 (ddd, J = 6.2, 5.0, 3.7 Hz, 6H), 2.19 (d, J = 6.6 Hz, 2H),1.89 (t, J = 5.2 Hz, 6H). 511

510.2 1H NMR (300 MHz, Chloroform-d) δ 8.63 (d, J = 1.9 Hz, 1H), 8.55(d, J = 1.9 Hz, 1H), 7.93 (d, J = 1.7 Hz, 1H), 6.85 (dd, J = 16.5, 2.5Hz, 2H), 4.74 (ddd, J = 21.7, 7.5, 3.9 Hz, 2H), 4.09 (p, J = 7.7, 7.2Hz, 1H), 3.92- 3.79 (m, 4H), 3.71 (dd, J = 5.7, 3.7 Hz, 4H), 3.57 (dd, J= 5.7, 3.7 Hz, 4H), 3.34- 3.20 (m, 4H), 2.15 (td, J = 11.0, 10.0, 6.2Hz, 2H), 1.94-1.79 (m, 6H). 512

468.27 1H NMR (300 MHz, Chloroform-d) δ 8.71 (d, J = 1.9 Hz, 1H), 8.64(d, J = 1.9 Hz, 1H), 7.99 (d, J = 1.8 Hz, 1H), 7.00-6.85 (m, 2H), 4.77(d, J = 6.6 Hz, 2H), 4.14 (q, J = 7.2 Hz, 1H), 4.03- 3.87 (m, 4H), 3.78-3.64 (m, 1H), 3.43- 3.28 (m, 4H), 3.15 (d, J = 2.5 Hz, 6H), 2.98- 2.84(m, 1H), 2.20 (d, J = 11.9 Hz, 2H), 1.94 (p, J = 5.8, 5.3 Hz, 6H). 525

379.26 1H NMR (400 MHz, CDCl3) δ 8.71 (d, J = 1.9 Hz, 1H), 8.63 (d, J =1.9 Hz, 1H), 8.31 (s, 1H), 8.30 (s, 1H), 6.93 (d, J = 2.4 Hz, 1H),6.64-6.54 (m, 2H), 5.38 (d, J = 5.1 Hz, 1H), 5.17-5.07 (m, 1H),4.68-4.56 (m, 1H), 3.95-3.85 (m, 4H), 3.35-3.27 (m, 4H), 2.94 (ddd, J =13.4, 8.2, 5.1 Hz, 2H), 2.59 (ddd, J = 13.9, 7.0, 4.5 Hz, 2H). 531

425.19 1H NMR (300 MHz, Chloroform-d) δ 8.71 (d, J = 1.9 Hz, 1H), 8.64(d, J = 1.9 Hz, 1H), 8.39 (dd, J = 2.7, 0.5 Hz, 1H), 8.04 (d, J = 3.4Hz, 1H), 6.97 (d, J = 2.5 Hz, 1H), 6.92 (d, J = 2.5 Hz, 1H), 5.15 (d, J= 8.1 Hz, 1H), 4.83 (dq, J = 5.1, 2.6 Hz, 1H), 4.25 (dd, J = 8.1, 4.6Hz, 1H), 3.98-3.88 (m, 4H), 3.39-3.28 (m, 4H), 2.34-2.17 (m, 2H),2.03-1.84 (m, 6H). 532

441.29 1H NMR (400 MHz, CDCl3) δ 8.69 (d, J = 1.9 Hz, 1H), 8.61 (d, J =1.9 Hz, 1H), 8.13 (d, J = 4.8 Hz, 1H), 6.96 (d, J = 2.4 Hz, 1H), 6.90(d, J = 2.5 Hz, 1H), 6.54 (d, J = 5.2 Hz, 1H), 5.36 (s, 1H), 4.79 (s,1H), 4.03 (s, 1H), 3.97-3.84 (m, 4H), 3.41-3.23 (m, 4H), 2.26-2.12 (m,2H), 1.98-1.81 (m, 6H). 533

441.25 1H NMR (400 MHz, CDCl3) δ 8.69 (d, J = 1.9 Hz, 1H), 8.60 (d, J =1.9 Hz, 1H), 7.99 (s, 1H), 6.96 (d, J = 2.4 Hz, 1H), 6.90 (d, J = 2.4Hz, 1H), 6.22 (d, J = 5.9 Hz, 1H), 5.22 (s, 1H), 4.81 (s, 1H), 4.03 (dt,J = 12.1, 6.2 Hz, 1H), 3.94-3.86 (m, 4H), 3.40-3.26 (m, 4H), 2.27-2.13(m, 2H), 1.90 (t, J = 17.4 Hz, 6H). 534

492.39 1H NMR (400 MHz, CDCl3) δ 8.69 (s, 1H), 8.61 (s, 1H), 7.89 (d, J= 5.5 Hz, 1H), 6.95 (s, 1H), 6.89 (s, 1H), 5.71 (d, J = 5.8 Hz, 1H),4.76 (s, 2H), 4.01- 3.81 (m, 5H), 3.74 (s, 8H), 3.41-3.26 (m, 4H),2.26-2.12 (m, 2H), 1.98-1.81 (m, 6H). 535

505.35 1H NMR (40 0MHz, CDCl3) δ 8.69 (d, J = 1.9 Hz, 1H), 8.61 (d, J =1.9 Hz, 1H), 7.88 (d, J = 5.7 Hz, 1H), 6.95 (d, J = 2.4 Hz, 1H), 6.89(d, J = 2.4 Hz, 1H), 5.67 (d, J= 5.8 Hz, 1H), 4.84-4.63 (m, 2H),3.99-3.83 (m, 5H), 3.82-3.73 (m, 4H), 3.37-3.27 (m, 4H), 2.49-2.40 (m,4H), 2.33 (s, 3H), 2.23-2.11 (m, 2H), 1.96-1.82 (m, 6H). 537

505.35 1H NMR (400 MHz, CDCl3) δ 8.69 (d, J = 1.7 Hz, 1H), 8.60 (d, J =1.7 Hz, 1H), 8.18 (s, 1H), 6.95 (d, J = 2.2 Hz, 1H), 6.90 (s, 1H), 5.42(s, 1H), 4.78 (s, 1H), 4.71 (d, J = 8.3 Hz, 1H), 3.98-3.87 (m, 4H), 3.83(s, 1H), 3.65-3.48 (m, 4H), 3.41-3.24 (m, 4H), 2.54-2.42 (m, 4H), 2.33(s, 3H), 2.25- 2.11 (m, 2H), 1.97- 1.82 (m, 6H). 539

481.3 1H NMR (300 MHz, CDCl3): ppm 1.76- 1.97 (m, 6 H), 2.15- 2.26 (m, 2H), 3.31- 3.35 (m, 4 H), 3.43 (s, 3 H), 3.61-3.72 (m, 3 H), 3.90-3.93(m, 4 H), 4.45-4.48 (m, 2 H), 4.76-4.82 (m, 1 H), 4.94 (d, J = 7.1 Hz, 1H), 5.73 (s, 1 H), 6.89 (d, J = 2. 547

505.4 1H NMR (400 MHz, CDCl3) δ 8.70 (d, J = 1.9 Hz, 1H), 8.63 (d, J =1.9 Hz, 1H), 7.90 (d, J = 6.0 Hz, 1H), 6.96 (d, J = 2.4 Hz, 1H), 6.92(d, J = 2.3 Hz, 1H), 5.89 (d, J = 6.1 Hz, 1H), 4.96 (s, 1H), 4.77 (s,1H), 4.08- 3.98 (m, 1H), 3.96- 3.87 (m, 4H), 3.70- 3.53 (m, 4H), 3.43-3.27 (m, 4H), 2.53- 2.42 (m, 4H), 2.35 (s, 3H), 2.25-2.13 (m, 2H),2.00-1.85 (m, 6H). 548

492.39 1H NMR (400 MHz, CDCl3) δ 8.70 (d, J = 1.8 Hz, 1H), 8.63 (d, J =1.9 Hz, 1H), 7.93 (d, J = 5.9 Hz, 1H), 6.96 (d, J = 2.3 Hz, 1H), 6.91(d, J = 2.3 Hz, 1H), 5.87 (d, J = 6.1 Hz, 1H), 4.97 (s, 1H), 4.77 (s,1H), 4.02 (s, 1H), 3.97-3.88 (m, 4H), 3.83-3.70 (m, 4H), 3.63-3.52 (m,4H), 3.39-3.27 (m, 4H), 2.25-2.12 (m, 2H), 2.01-1.85 (m, 6H). 553

480.19 1H NMR (300 MHz, Chloroform-d) δ 8.71 (d, J = 1.9 Hz, 1H), 8.63(d, J = 1.9 Hz, 1H), 6.93 (dd, J = 17.8, 2.5 Hz, 2H), 5.07 (s, 1H), 4.78(d, J = 6.2 Hz, 1H), 4.68 (d, J = 7.9 Hz, 1H), 4.03- 3.90 (m, 4H), 3.87(s, 3H), 3.71 (s, 1H), 3.42- 3.28 (m, 4H), 3.13 (s, 6H), 2.27-2.00 (m,2H), 2.00-1.79 (m, 6H). 558

481.3 1H NMR (300 MHz, CDCl3): ppm 1.84- 1.99 (m, 6H), 2.11- 2.25 (m, 2H), 3.32- 3.36 (m, 4 H), 3.43 (s, 3 H), 3.70-3.73 (m, 2 H), 3.90-3.93(m, 4 H), 3.96-4.07 (m, 1 H), 4.41-4.44 (m, 2 H), 4.72-4.80 (m, 1 H),5.10 (br. s, 1 H), 6.06 (d, J = 5.6 565

479.1 1H NMR (300 MHz, Chloroform-d) δ 8.55 (d, J = 1.9 Hz, 1H), 8.47(d, J = 1.9 Hz, 1H), 6.93 (s, 1H), 6.86- 6.72 (m, 2H), 5.25 (s, 1H),4.66 (s, 1H), 4.05- 3.90 (m, 1H), 3.86- 3.71 (m, 7H), 3.30- 3.09 (m,4H), 2.27 (d, J = 2.2 Hz, 3H), 2.04 (d, J = 9.2 Hz, 2H), 1.75 (d, J =4.8 Hz, 6H). 567

504.1 1H NMR (300 MHz, Chloroform-d) δ 8.70 (d, J = 1.9 Hz, 1H), 8.63(d, J = 1.9 Hz, 1H), 7.74 (s, 1H), 7.00- 6.89 (m, 2H), 4.91 (s, 1H),4.76 (d, J = 6.1 Hz, 1H), 4.04-3.93 (m, 5H), 3.43-3.28 (m, 4H),2.26-2.14 (m, 2H), 2.08 (d, J = 0.8 Hz, 3H), 2.00- 1.92 (m, 6H), 1.69(d, J = 21.2 Hz, 10H). 572

472.2 1H NMR (300 MHz, CDCl3): ppm 1.82- 1.99 (m, 6 H), 2.14- 2.28 (m, 2H), 3.32- 3.35 (m, 4 H), 3.80- 3.88 (m, 1 H), 3.90- 3.93 (m, 4H), 4.70-4.83 (m, 2 H), 6.37 (d, J = 8.8 Hz, 1 H), 6.38 (t, J = 73.8 Hz, 1 H),6.90 (d, J = 2.3 Hz, 1 H), 6.95 (d, J = 573

464.3 1H NMR (300 MHz, CDCl3): ppm 1.52 (s, 6 H), 1.73 (br s, 1 H),1.85-1.96 (m, 6 H), 2.11-2.25 (m, 2 H), 3.30-3.36 (m, 4 H), 3.88-4.00(m, 5 H), 4.55 (d, J = 8.1 Hz, 1 H), 4.74-4.81 (m, 1 H), 6.52 (s, 1 H),6.57 (dd, J = 5.4, 1.4 Hz, 1 H), 6.90 (d, 574

437.42 1H NMR (400 MHz, CDCl3) δ 8.69 (d, J = 1.9 Hz, 1H), 8.61 (d, J =1.9 Hz, 1H), 7.99 (s, 2H), 6.95 (d, J = 2.4 Hz, 1H), 6.89 (d, J = 2.4Hz, 1H), 4.78 (s, 1H), 4.01-3.84 (m, 7H), 3.41 (s, 2H), 3.37- 3.27 (m,4H), 2.26- 2.14 (m, 2H), 1.93- 1.81 (m, 6H). 575

407.35 1H NMR (400 MHz, CDCl3) δ 8.69 (d, J = 1.9 Hz, 1H), 8.62 (d, J=1.9 Hz, 1H), 8.57 (s, 1H), 8.13 (s, 2H), 6.98 (d, J = 2.4 Hz, 1H), 6.91(d, J = 2.5 Hz, 1H), 4.86-4.73 (m, 1H), 3.95-3.88 (m, 4H), 3.56-3.49 (m,1H), 3.39-3.31 (m, 4H), 2.30-2.18 (m, 2H), 1.97-1.84 (m, 6H). 576

450.13 1H NMR (300 MHz, Chloroform-d) δ 8.71 (d, J = 2.0 Hz, 1H), 8.63(d, J = 1.9 Hz, 1H), 8.13 (s, 1H), 7.01- 6.87 (m, 2H), 4.81 (s, 1H),4.57 (s, 2H), 4.06 (s, 1H), 3.99-3.87 (m, 4H), 3.35 (dd, J = 5.9, 3.8Hz, 4H), 2.45 (s, 3H), 2.20 (d, J = 8.0 Hz, 2H), 1.93 (d, J = 5.1 Hz,6H). 577

437.25 1H NMR (300 MHz, Chloroform-d) δ 8.71 (d, J = 1.9 Hz, 1H), 8.63(d, J = 2.0 Hz, 1H), 8.02 (d, J = 1.4 Hz, 1H), 7.87 (d, J = 1.5 Hz, 1H),6.94 (dd, J = 16.2, 2.5 Hz, 2H), 4.89-4.71 (m, 2H), 4.65 (s, 2H), 4.06-3.85 (m, 4H), 3.40- 3.27 (m, 4H), 2.23 (dt, J = 11.5, 5.5 Hz, 2H),2.04-1.80 (m, 6H). 579

505.53 1H NMR (400 MHz, CDCl3) δ 8.68 (s, 1H), 8.61 (s, 1H), 8.07 (s,2H), 6.94 (s, 1H), 6.90 (s, 1H), 4.96 (d, J = 8.1 Hz, 1H), 4.78 (s, 1H),4.02-3.81 (m, 5H), 3.47-3.19 (m, 5H), 3.19-2.83 (m, 4H), 2.76-2.52 (m,3H), 2.37 (s, 3H), 2.26- 2.11 (m, 2H), 2.03- 1.82 (m, 6H). 582

450.3 1H NMR (300 MHz, CDCl3): ppm 1.83 ñ 1.96 (m, 6 H), 2.14- 2.26 (m,2 H), 3.30- 3.36 (m, 4 H), 3.41 (s, 3 H), 3.87 ñ 3.95 (m, 5 H), 4.36 (s,2 H), 4.59- 4.89 (m, 2 H), 6.38 (s, 1 H), 6.48 (d, J = 5.2 Hz, 1H), 6.90(d, J = 2.4 Hz, 1H), 6.95 (d, J = 2.4 583

466.3 1H NMR (300 MHz, CDCl3): ppm 1.84- 2.00 (m, 6 H), 2.09- 2.24 (m, 2H), 3.30- 3.37 (m, 4 H), 3.64- 3.74 (m, 1 H), 3.78 (s, 3H), 3.89-3.95(m, 7 H), 4.71-4.79 (m, 1 H), 5.88 (d, J = 8.3 Hz, 1H), 6.90 (d, J = 2.4Hz, 1H), 6.95 (d, J = 2.4 Hz, 1 H) 584

436.3 1H NMR (300 MHz, CDCl3): ppm 1.86- 2.04 (m, 6 H), 2.12- 2.26 (m, 2H), 3.28- 3.39 (m, 4 H), 3.88- 3.96 (m, 4 H), 4.26 (br s, 1 H), 4.62 (s,2 H), 4.69-4.78 (m, 1 H), 5.56 (br s, 1 H), 6.50 (t, J = 5.3 Hz, 1 H),6.90 (d, J = 1.9 Hz, 1 H), 6.93 (d, J 586

420.3 1H NMR (300 MHz, CDCl3): ppm 1.88 ñ 2.01 (m, 6 H), 2.10 (s, 3 H),2.15-2.25 (m, 2 H), 3.30-3.37 (m, 4 H), 3.89 ñ 3.95 (m, 4 H), 4.25 (brs, 2 H), 4.71-4.78 (m, 1 H), 6.51 (dd, J = 6.8, 5.4 Hz, 1 H), 6.90 (d, J= 2.3 Hz, 1 H), 6.94 (d, J = 2.3 Hz, 1H 587

474.2 1H NMR (300 MHz, CDCl3): ppm 1.82- 1.98 (m, 6 H), 2.12- 2.26 (m, 2H), 3.25- 3.43 (m, 4 H), 3.82- 3.98 (m, 5 H), 4.80 (br s, 2 H), 6.52 (d,J = 8.4 Hz, 1 H), 6.87- 6.93 (m, 2 H), 6.95- 7.00 (m, 1 H), 7.50 (t, J =7.9 Hz, 1 H), 8.63 (d, J = 1.2 Hz, 1H 588

450.3 1H NMR (300 MHz, CDCl3): ppm 1.38 (t, J = 7.0 Hz, 3 H), 1.83- 2.00(m, 6 H), 2.11- 2.24 (m, 2 H), 3.30- 3.37 (m, 4 H), 3.75- 3.85 (m, 1 H),3.89- 3.94 (m, 4 H), 4.25 (q, J = 7.0 Hz, 2 H), 4.42- 4.54 (m, 1 H),4.72- 4.79 (m, 1 H), 5.94 (d, J = 7.7 H 589

474.2 1H NMR (300 MHz, CDCl3): ppm 1.86- 1.99 (m, 6 H), 2.16- 2.27 (m, 2H), 3.31- 3.36 (m, 4 H), 3.88- 3.95 (m, 5 H), 4.77- 4.84 (m, 1 H), 4.90(br s, 1 H), 6.55 (s, 1 H), 6.72 (d, J = 5.0 Hz, 1 H), 6.91 (d, J = 2.4Hz, 1 H), 6.95 (d, J = 2.4 Hz, 1 H), 8. 590

491.3 1H NMR (300 MHz, CDCl3): ppm 1.82- 1.98 (m, 6 H), 2.10- 2.22 (m, 2H), 3.31- 3.35 (m, 4 H), 3.41- 3.45 (m, 4 H), 3.69- 3.82 (m, H), 3.90-3.93 (m, 4 H), 4.43 (d, J = 7.7 Hz, 1 H), 4.70- 4.77 (m, 1 H), 5.81 (d,J = 8.0 Hz, 1 H), 5.90 (d, J = 8.0 H 591

424.2 1H NMR (300 MHz, CDCl3): ppm 1.88- 2.00 (m, 6 H), 2.13- 2.28 (m, 2H), 3.29- 3.38 (m, 4 H), 3.88- 3.95 (m, 4 H), 4.21 (br s, 1 H),4.62-4.84 (m, 2 H), 6.45-6.54 (m, 1 H), 6.90 (d, J = 2.4 Hz, 1 H), 6.95(d, J = 2.4 Hz, 1 H), 7.07- 7.18 (m, 1 H). 592

436.3 1H NMR (300 MHz, CDCl3): ppm 1.84- 2.03 (m, 6 H), 2.11- 2.24 (m, 2H), 3.30- 3.37 (m, 4 H), 3.84 (s, 3H), 3.89-3.95 (m, 4 H), 4.17-4.29 (m,1 H), 4.67-4.76 (m, 1 H), 5.08 (d, J = 7.4 Hz, 1 H), 6.50 (dd, J = 7.3,5.2 Hz, 1 H), 6.82 (d, J = 7.4 Hz 593

474.2 1H NMR (300 MHz, CDCl3): ppm 1.86- 2.00 (m, 6 H), 2.13- 2.26 (m, 2H), 3.30- 3.38 (m, 4 H), 3.88- 3.97 (m, 4 H), 4.22- 4.35 (m, 1 H), 4.73-4.80 (m, 1 H), 4.89- 4.99 (m, 1 H), 6.60 (dd, J = 7.1, 5.1 Hz, 1 H),6.91 (d, J = 2.3 Hz, 1 H), 6.96 (d, J = 594

442.2 1H NMR (300 MHz, CDCl3): ppm 1.85- 2.00 (m, 6 H), 2.11- 2.28 (m, 2H), 3.29- 3.39 (m, 4 H), 3.87- 3.97 (m, 4 H), 4.06- 4.18 (m, 1 H), 4.56(d, J = 7.6 Hz, 1H), 4.73- 4.81 (m, 1 H), 6.90 (d, J = 2.4 Hz, 1 H),6.95 (d, J = 2.4 Hz, 1 H), 7.05 (ddd, J = 595

434.3 1H NMR (300 MHz, CDCl3): ppm 1.88- 2.02 (m, 6 H), 2.08 (s, 3 H),2.15 (s, 3 H), 2.14-2.24 (m, 2 H), 3.31-3.36 (m, 4 H), 3.89-3.95 (m, 4H), 4.03-4.16 (m, 1 H), 4.23 (br s, 1H), 4.69- 4.77 (m, 1 H), 6.90 (d, J= 2.4 Hz, 1 H), 6.94 (d, J = 2.4 Hz 597

449.23 1H NMR (300 MHz, Chloroform-d) δ 8.71 (d, J = 1.9 Hz, 1H), 8.63(d, J = 1.9 Hz, 1H), 7.07-6.87 (m, 2H), 6.02 (s, 1H), 4.83 (d, J = 5.5Hz, 1H), 4.03-3.81 (m, 5H), 3.44-3.25 (m, 4H), 2.72-2.57 (m, 1H), 2.51(s, 3H), 2.29- 2.11 (m, 2H), 2.05- 1.72 (m, 6H), 1.27 (t, J = 7.6 Hz,3H). 598

450.23 1H NMR (300 MHz, Chloroform-d) δ 8.61 (d, J = 1.9 Hz, 1H), 8.54(d, J = 1.9 Hz, 1H), 6.93-6.72 (m, 3H), 6.55 (s, 1H), 4.71 (s, 1H), 4.01(s, 1H), 3.92-3.76 (m, 4H), 3.34-3.18 (m, 4H), 2.98 (s, 6H), 2.07 (d, J= 24.1 Hz, 2H), 1.87 (d, J = 9.2 Hz, 6H). 600

464.26 1H NMR (300 MHz, Chloroform-d) δ 8.71 (d, J = 2.0 Hz, 1H), 8.62(d, J = 2.0 Hz, 1H), 7.00-6.85 (m, 2H), 5.44 (d, J = 8.5 Hz, 1H), 4.78(s, 1H), 3.93 (t, J = 4.8 Hz, 4H), 3.35 (t, J = 4.9 Hz, 4H), 3.06-2.91(m, 1H), 2.15 (d, J = 26.6 Hz, 2H), 1.89 (s, 6H), 1.30-0.94 (m, 4H). 601

461.32 1H NMR (300 MHz, Chloroform-d) δ 8.70 (d, J = 1.9 Hz, 1H), 8.62(d, J = 1.9 Hz, 1H), 7.06 (d, J = 9.1 Hz, 1H), 6.98-6.86 (m, 2H), 6.58(d, J = 9.1 Hz, 1H), 4.81 (dq, J = 5.5, 2.7 Hz, 1H), 4.52 (s, 1H), 4.17(d, J = 5.0 Hz, 1H), 4.01- 3.87 (m, 4H), 3.67 (dq, J = 8.9, 8.1 Hz, 1H),3.43-3.30 (m, 4H), 2.47-2.29 (m, 4H), 2.22 (dt, J = 11.9, 5.5 Hz, 2H),2.05-1.82 (m, 6H). 602

464.1 1H NMR (300 MHz, Chloroform-d) δ 8.70 (d, J = 1.9 Hz, 1H), 8.62(d, J = 1.9 Hz, 1H), 7.04-6.92 (m, 2H), 4.82 (d, J = 3.0 Hz, 1H), 3.93(dd, J = 5.9, 3.8 Hz, 4H), 3.65 (d, J = 8.7 Hz, 1H), 3.44-3.29 (m, 4H),3.08 (s, 6H), 2.39 (s, 3H), 2.21 (d, J = 14.4 Hz, 2H), 1.92 (dt, J =17.0, 10.7 Hz, 6H). 603

450.1 1H NMR (300 MHz, Chloroform-d) δ 8.70 (d, J = 1.9 Hz, 1H), 8.62(d, J = 1.9 Hz, 1H), 8.18 (s, 1H), 7.02- 6.86 (m, 2H), 4.81 (s, 2H),4.00-3.76 (m, 5H), 3.42-3.26 (m, 4H), 3.07 (s, 6H), 2.20 (q, J = 6.3,5.8 Hz, 2H), 2.01-1.79 (m, 6H). 604

452.1 1H NMR (300 MHz, Chloroform-d) δ 8.62 (d, J = 1.9 Hz, 1H), 8.53(d, J = 1.9 Hz, 1H), 6.87 (d, J = 2.5 Hz, 1H), 6.81 (d, J = 2.6 Hz, 1H),4.71 (s, 1H), 3.94-3.72 (m, 7H), 3.25 (dd, J = 5.8, 3.8 Hz, 4H), 2.29(d, J = 10.4 Hz, 3H), 2.21- 2.02 (m, 2H), 1.93- 1.71 (m, 6H). 605

482.3 607

469.08 1H NMR (300 MHz, Chloroform-d) δ 8.71 (d, J = 1.9 Hz, 1H), 8.63(d, J = 1.9 Hz, 1H), 7.79 (d, J = 2.9 Hz, 1H), 6.93 (dd, J = 17.8, 2.5Hz, 2H), 5.19- 5.07 (m, 1H), 4.80 (dt, J = 6.7, 3.4 Hz, 1H), 4.32 (q, J= 7.1 Hz, 3H), 3.98-3.87 (m, 4H), 3.41-3.29 (m, 4H), 2.30-2.13 (m, 2H),2.03-1.84 (m, 6H), 1.41 (t, J = 7.1 Hz, 3H). 608

468.63 1H NMR (300 MHz, Chloroform-d) δ 8.71 (d, J = 1.9 Hz, 1H), 8.63(d, J = 2.0 Hz, 1H), 6.94 (dd, J = 18.9, 2.5 Hz, 2H), 5.42 (d, J = 8.4Hz, 1H), 4.79 (s, 1H), 4.22- 4.06 (m, 1H), 4.03- 3.89 (m, 10H), 3.42-3.22 (m, 4H), 2.19 (m, 2H), 2.01-1.79 (m, 6H). 609

471.87 1H NMR (300 MHz, Chloroform-d) δ 8.72 (d, J = 1.9 Hz, 1H), 8.63(d, J = 1.9 Hz, 1H), 7.03-6.84 (m, 2H), 5.66 (d, J = 9.1 Hz, 1H), 4.82(s, 1H), 4.09-3.84 (m, 7H), 3.35 (dd, J = 5.5, 3.6 Hz, 4H), 2.20 (s,2H), 1.91 (d, J = 6.4 Hz, 6H). 612

481.3 1H NMR (300 MHz, CDCl3): ppm 1.83- 1.96 (m, 6 H), 2.14- 2.24 (m, 2H), 3.31- 3.35 (m, 4 H), 3.44 (s, 3 H), 3.69-3.72 (m, 2 H), 3.89-3.95(m, 5 H), 4.06-4.09 (m, 2 H), 4.74-4.83 (m, 1 H), 5.04-5.15 (m, 1 H),6.90 (d, J = 2.4 Hz, 1 H), 6.94 (d, 614

424.2 1H NMR (300 MHz, CDCl3): ppm 1.83- 1.95 (m, 6 H), 2.12- 2.26 (m, 2H), 3.30- 3.40 (m, 4 H), 3.85- 3.96 (m, 5 H), 4.55- 4.68 (m, 1 H), 4.74-4.82 (m, 1 H), 6.10 (dd, J = 7.7, 1.8 Hz, 1 H), 6.18 (dd, J = 8.0, 1.8Hz, 1 H), 6.90 (d, J = 1.8 Hz, 1 H), 6 615

436.3 1H NMR (300 MHz, CDCl3): ppm 1.82- 2.10 (m, 7 H), 2.14- 2.29 (m, 2H), 3.26- 3.40 (m, 4 H), 3.82- 4.05 (m, 5 H), 4.64 (s, 2 H), 4.75-4.84(m, 1 H), 4.85-5.13 (m, 1 H), 6.46 (s, 1H), 6.51 (d, J = 5.3 Hz, 1 H),6.90 (d, J = 1.7 Hz, 1 H), 6.94 (d, 616

465.1 1H NMR (300 MHz, Chloroform-d) δ 8.71 (d, J = 1.9 Hz, 1H), 8.63(d, J = 1.9 Hz, 1H), 6.95 (dd, J = 16.6, 2.5 Hz, 2H), 6.30 (s, 1H), 5.02(s, 1H), 4.82 (s, 1H), 4.38 (d, J = 0.9 Hz, 2H), 3.99- 3.83 (m, 5H),3.39- 3.24 (m, 4H), 2.49 (s, 3H), 2.33-2.11 (m, 2H), 1.91 (q, J = 9.0,6.9 Hz, 6H). 618

463.13 1H NMR (300 MHz, Chloroform-d) δ 8.63 (d, J = 1.9 Hz, 1H), 8.55(d, J = 1.9 Hz, 1H), 6.86 (dd, J = 16.5, 2.5 Hz, 2H), 5.94 (s, 1H), 4.73(s, 1H), 3.85 (dd, J = 5.9, 3.7 Hz, 4H), 3.35-3.20 (m, 4H), 2.67-2.51(m, 2H), 2.26 (s, 3H), 2.09 (d, J = 36.8 Hz, 3H), 1.94-1.59 (m, 6H),0.91 (t, J = 7.4 Hz, 3H). 622

424.2 1H NMR (300 MHz, CDCl3): ppm 1.81- 1.98 (m, 6 H), 2.13- 2.25 (m, 2H), 3.29- 3.38 (m, 4 H), 3.75- 3.85 (m, 1 H), 3.88- 3.96 (m, 4 H), 4.56-4.71 (m, 1 H), 4.75- 4.83 (m, 1 H), 6.36 (dd, J = 9.1, 3.3 Hz, 1 H),6.90 (d, J = 1.9 Hz, 1 H), 6.95 (d, J = 623

420.3 1H NMR (300 MHz, CDCl3): ppm 1.80- 1.98 (m, 6 H), 2.13- 2.23 (m, 2H), 2.38 (s, 3 H), 3.25-3.37 (m, 4 H), 3.62-3.77 (m, 1 H), 3.88-3.97 (m,4 H), 4.76-4.83 (m, 1 H), 6.23 (d, J = 7.9 Hz, 1 H), 6.43 (d, J = 7.3Hz, 1H), 6.91 (d, J = 2.5 Hz, 1 H) 625

480.3 1H NMR (300 MHz, CDCl3): ppm 1.81- 1.95 (m, 6 H), 2.11- 2.26 (m, 2H), 3.31- 3.35 (m, 4 H), 3.44 (s, 3 H), 3.69-3.73 (m, 2 H), 3.76-3.84(m, 1 H), 3.89-3.93 (m, 4 H), 4.04-4.08 (m, 2 H), 4.44 (br s, 1 H),4.74-4.81 (m, 1 H), 6.37 (d, J = 8.9 626

504.3 1H NMR (300 MHz, CDCl3): ppm 1.81- 1.98 (m, 6 H), 2.12- 2.25 (m, 2H), 2.37- 2.48 (m, 3 H), 2.58- 2.77 (m, 4 H), 3.04- 3.16 (m, 4 H), 3.28-3.37 (m, 4 H), 3.77- 3.85 (m, 1 H), 3.87- 3.97 (m, 4 H), 4.32- 4.51 (m,1 H), 4.73- 4.83 (m, 1 H), 6.38 ( 627

436.3 1H NMR (300 MHz, CDCl3): ppm 1.81- 2.03 (m, 7 H), 2.12- 2.28 (m, 2H), 3.28- 3.39 (m, 4 H), 3.81 (s, 3 H), 3.87-3.98 (m, 5 H), 4.76-4.84(m, 1 H), 5.87 (d, J = 1.5 Hz, 1 H), 6.21 (dd, J = 5.9, 1.5 Hz, 1 H),6.91 (d, J = 1.7 Hz, 1 H), 6.95 (d, J = 1 629

518.4 1H NMR (300 MHz, CDCl3) δ 8.62 (s, 1H), 8.54 (s, 1H), 8.15- 7.93(m, 1H), 7.40 (d, J = 8.0 Hz, 1H), 6.88 (s, 1H), 6.84 (s, 1H), 6.34 (d,J = 8.6 Hz, 1H), 4.83-4.56 (m, 2H), 4.40-4.22 (m, 1H), 4.00-3.62 (m,5H), 3.47-3.04 (m, 7H), 2.24-2.02 (m, 2H), 1.96-1.70 (m, 6H). 630

504.22 1H NMR (400 MHz, CDCl3) δ 8.68 (d, J = 1.8 Hz, 1H), 8.59 (d, J =1.8 Hz, 1H), 8.06 (s, 1H), 7.55 (d, J = 8.3 Hz, 1H), 6.94 (d, J = 2.3Hz, 1H), 6.89 (d, J = 2.2 Hz, 1H), 6.41 (d, J = 8.7 Hz, 1H), 4.88 (dd, J= 13.4, 6.7 Hz, 1H), 4.85-4.71 (m, 2H), 3.98-3.85 (m, 5H), 3.39-3.28 (m,4H), 2.25-2.12 (m, 2H), 1.94-1.82 (m, 6H). 633

435.2 1H NMR (300 MHz, Chloroform-d) δ 8.70 (d, J = 1.9 Hz, 1H), 8.63(d, J = 1.9 Hz, 1H), 8.18 (d, J = 5.1 Hz, 1H), 7.01-6.88 (m, 2H), 6.42(d, J = 5.1 Hz, 1H), 5.15 (d, J = 8.0 Hz, 1H), 4.79 (d, J = 5.7 Hz, 1H),4.17- 4.00 (m, 1H), 3.98- 3.83 (m, 4H), 3.67 (td, J = 6.7, 4.0 Hz, 1H),3.42-3.31 (m, 4H), 2.59 (q, J = 7.6 Hz, 2H), 2.20 (q, J = 6.1 Hz, 2H),2.03-1.86 (m, 6H), 1.26 (t, J = 7.6 Hz, 3H). 634

449.2 1H NMR (300 MHz, Chloroform-d) δ 8.71 (d, J = 1.9 Hz, 1H), 8.63(d, J = 1.9 Hz, 1H), 8.19 (d, J = 5.1 Hz, 1H), 7.01-6.88 (m, 2H), 6.42(d, J = 5.2 Hz, 1H), 5.14 (d, J = 7.9 Hz, 1H), 4.80 (dt, J = 7.8, 3.8Hz, 1H), 4.06 (d, J = 4.1 Hz, 0H), 3.99-3.85 (m, 4H), 3.46-3.24 (m, 4H),2.78 (hept, J = 7.0 Hz, 1H), 2.34- 2.11 (m, 2H), 2.04- 1.81 (m, 7H),1.25 (d, J = 7.0 Hz, 6H). 636

461.1 1H NMR (300 MHz, Chloroform-d) δ 8.63 (d, J = 1.9 Hz, 1H), 8.54(d, J = 1.9 Hz, 1H), 6.86 (dd, J = 16.2, 2.5 Hz, 2H), 5.85 (s, 1H), 4.75(d, J = 5.5 Hz, 1H), 3.96- 3.76 (m, 4H), 3.34- 3.19 (m, 4H), 2.41 (s,3H), 2.13 (d, J = 8.5 Hz, 2H), 1.82 (q, J = 10.2, 8.8 Hz, 6H), 1.58-1.33 (m, 3H), 1.03- 0.86 (m, 3H). 641

371.56 1H NMR (400 MHz, CDCl3) δ 8.73-8.65 (m, 1H), 8.60 (d, J = 1.8 Hz,1H), 6.93 (d, J = 2.4 Hz, 1H), 6.89 (d, J = 2.3 Hz, 1H), 4.75 (s, 1H),3.95-3.87 (m, 4H), 3.39-3.28 (m, 4H), 3.05-2.93 (m, 1H), 2.82-2.69 (m,1H), 2.24-2.08 (m, 2H), 1.80-1.71 (m, 6H), 1.07 (d, J = 6.2 Hz, 6H). 642

424.53 1H NMR (400 MHz, CDCl3) δ 8.70 (d, J = 1.9 Hz, 1H), 8.61 (d, J =1.9 Hz, 1H), 7.78 (d, J = 5.8 Hz, 1H), 6.96 (d, J = 2.4 Hz, 1H), 6.90(d, J = 2.4 Hz, 1H), 6.36-6.26 (m, 1H), 5.99 (d, J = 1.9 Hz, 1H), 4.79(d, J = 2.4 Hz, 1H), 4.47 (d, J = 7.1 Hz, 1H), 3.99- 3.85 (m, 4H), 3.56-3.49 (m, 1H), 3.39- 3.26 (m, 4H), 2.28- 2.16 (m, 2H), 1.98- 1.80 (m,6H). 643

492.2 1H NMR (300 MHz, Chloroform-d) δ 8.70 (d, J = 1.9 Hz, 1H), 8.61(d, J = 1.9 Hz, 1H), 8.57-8.45 (m, 1H), 7.81 (d, J = 8.4 Hz, 1H), 7.18(d, J = 1.2 Hz, 1H), 6.93 (dd, J = 16.8, 2.5 Hz, 2H), 5.48 (s, 1H), 4.83(dp, J = 4.5, 2.4 Hz, 1H), 4.23 (dp, J = 8.3, 6.6 Hz, 1H), 4.00-3.85 (m,4H), 3.42-3.23 (m, 4H), 2.35-3.15 (m, 2H), 1.97-1.76 (m, 6H), 1.26 (d, J= 6.6 Hz, 6H). 644

465.16 1H NMR (300 MHz, Chloroform-d) δ 8.69 (d, J = 1.9 Hz, 1H), 8.61(d, J = 1.9 Hz, 1H), 6.93 (dd, J = 17.5, 2.5 Hz, 2H), 5.47 (s, 1H), 4.90(s, 1H), 4.31 (q, J = 7.1 Hz, 2H), 3.92 (dd, J = 5.9, 3.7 Hz, 4H), 3.60(s, 1H), 3.38-3.28 (m, 4H), 2.40 (s, 3H), 2.19 (d, J = 9.4 Hz, 2H),1.97-1.80 (m, 6H), 1.36 (t, J = 7.1 Hz, 3H), 4.83-4.76 (m, 1H). 650

447.2 1H NMR (300 MHz, Chloroform-d) δ 8.70 (d, J = 1.9 Hz, 1H), 8.62(d, J = 1.9 Hz, 1H), 8.47-8.36 (m, 1H), 6.96 (d, J = 2.5 Hz, 1H), 6.90(d, J = 2.5 Hz, 1H), 6.18 (d, J = 1.2 Hz, 1H), 4.95- 4.73 (m, 2H), 4.04-3.78 (m, 5H), 3.44- 3.24 (m, 4H), 2.30- 2.12 (m, 2H), 2.03- 1.70 (m,7H), 1.17- 0.80 (m, 4H). 651

435.18 1H NMR (300 MHz, Chloroform-d) δ 8.70 (d, J = 1.9 Hz, 1H), 8.61(d, J = 1.9 Hz, 1H), 8.50 (d, J = 1.1 Hz, 1H), 6.93 (dd, J = 15.9, 2.5Hz, 2H), 6.17 (d, J = 1.1 Hz, 1H), 4.98 (d, J = 8.1 Hz, 1H), 4.86-4.75(m, 1H), 3.97-3.87 (m, 4H), 3.39-3.29 (m, 4H), 2.61 (q, J = 7.6 Hz, 2H),2.21 (dt, J = 11.2, 5.0 Hz, 2H), 2.04- 1.82 (m, 6H), 1.25 (t, J = 7.6Hz, 3H). 652

449.23 1H NMR (300 MHz, Chloroform-d) δ 8.70 (d, J = 1.9 Hz, 1H), 8.61(d, J = 1.9 Hz, 1H), 8.52 (d, J = 1.1 Hz, 1H), 6.93 (dd, J = 16.0, 2.5Hz, 2H), 6.19- 6.12 (m, 1H), 4.91 (d, J = 8.0 Hz, 1H), 4.81 (d, J = 5.4Hz, 1H), 4.13-3.77 (m, 5H), 3.40-3.29 (m, 4H), 2.80 (hept, J = 6.8 Hz,1H), 2.27- 2.00 (m, 2H), 1.99- 1.83 (m, 6H), 1.40- 1.12 (m, 6H). 653

451.07 1H NMR (300 MHz, Methanol-d4/CDCl3) δ 8.82-8.68 (m, 2H), 8.61 (d,J = 0.8 Hz, 1H), 7.38 (t, J = 0.8 Hz, 1H), 7.18 (s, 1H), 5.11-4.99 (m,1H), 4.47-4.31 (m, 1H), 3.99-3.88 (m, 4H), 3.74-3.58 (m, 4H), 2.37-2.23(m, 2H), 2.13-1.80 (m, 6H). 658

450.3 1H NMR (300 MHz, CDCl3): ppm 1.37 (t, J = 7.0 HZ, 3 H), 1.84- 1.94(m, 6 H), 2.10- 2.25 (m, 2 H), 3.29- 3.38 (m, 4 H), 3.75- 3.85 (m, 1 H),3.88- 3.95 (m, 4 H), 3.97 (q, J = 7.0 Hz, 2 H), 4.22- 4.34 (m, 1 H),4.73- 4.80 (m, 1 H), 6.36 (d, J = 9.0 H 659

451.34 1H NMR (300 MHz, Chloroform-d) δ 8.69 (d, J = 1.9 Hz, 1H), 8.60(d, J = 1.9 Hz, 1H), 6.92 (dd, J = 19.6, 2.5 Hz, 2H), 5.25 (s, 1H), 4.76(q, J = 8.3, 7.2 Hz, 2H), 3.97- 3.87 (m, 4H), 3.58- 3.28 (m, 8H), 2.40(s, 3H), 2.24-2.13 (m, 2H), 2.07-1.78 (m, 6H). 660

661

662 663 664 665

666

Biological Assay of Compounds of the Invention EXAMPLE 11 DNA-PKInhibition Assay

Compounds were screened for their ability to inhibit DNA-PK kinase usinga standard radiometric assay. Briefly, in this kinase assay the transferof the terminal ³³P-phosphate in ³³P-ATP to a peptide substrate isinterrogated. The assay was carried out in 384-well plates to a finalvolume of 50 μL per well containing approximately 6 nM DNA-PK, 50 mMHEPES (pH 7.5), 10 mM MgCl₂, 25 mM NaCl, 0.01% BSA, 1 mM DTT, 10 μg/mLsheared double-stranded DNA (obtained from Sigma), 0.8 mg/mL DNA-PKpeptide(Glu-Pro-Pro-Leu-Ser-Gln-Glu-Ala-Phe-Ala-Asp-Leu-Trp-Lys-Lys-Lys,obtained from American Peptide), and 100 μM ATP.

Accordingly, compounds of the invention were dissolved in DMSO to make10 mM initial stock solutions. Serial dilutions in DMSO were then madeto obtain the final solutions for the assay. A 0.75 μL aliquot of DMSOor inhibitor in DMSO was added to each well, followed by the addition ofATP substrate solution containing ³³P-ATP (obtained from Perkin Elmer).The reaction was started by the addition of DNA-PK, peptide and ds-DNA.After 45 min, the reaction was quenched with 25 μL of 5% phosphoricacid. The reaction mixture was transferred to MultiScreen HTS 384-wellPH plates (obtained from Millipore), allowed to bind for one hour, andwashed three times with 1% phosphoric acid. Following the addition of 50μL of Ultima Gold™ high efficiency scintillant (obtained from PerkinElmer), the samples were counted in a Packard TopCount NXT MicroplateScintillation and Luminescence Counter (Packard BioScience). The K_(i)values were calculated using Microsoft Excel Solver macros to fit thedata to the kinetic model for competitive tight-binding inhibition.

Each of compounds 18, 23, 24, 27-30, 32-34, 36-42, 44-46, 49-55, 59-61,63-67, 69, 71, 72, 84, 87, 93, 94, 104, 108, 109, 134, 135, 139, 140,142-146, 152, 155, 158, 160-162, 164, 187, 189-191, 193-210, 215, 219,223-227, 233-237, 241-243, 245-247, 254, 256, 257, 260, 263-265,267-269, 272, 273, 275-277, 282, 283, 285, 286, 287, 291, 295, 297, 298,308, 309, 312, 314, 315-319, 321, 323, 324, 333-336, 340, 341, 351-354,366, 369, 373-377, 380, 382, 386-388, 393-397, 399, 401, 402, 404-409,417-422, 424, 427-430, 436, 438-451, 453-457, 463, 465, 467, 469, 471,472, 474, 476-478, 481, 482, 485, 486, 489-492, 494, 499, 511, 512, 525,531-535, 537, 539, 547, 548, 553, 558, 565, 567, 572-577, 579, 582-584,586-595, 597, 598, 600-605, 607-609, 612, 614-616, 618, 622, 623,625-627, 629, 630, 633, 634, 636, 642-644, 650-653, and 658-666 has aK_(i) of less than 1.0 micromolar for the inhibition of DNA-PK. Each ofcompounds 18, 23, 24, 27-30, 32-34, 36-37, 39-41, 44-46, 49-55, 59-61,63-67, 69, 71, 72, 84, 87, 93, 94, 104, 108, 109, 134, 135, 139, 140,142-146, 152, 155, 158, 160-162, 164, 187, 189-191, 193-200, 202-210,215, 219, 223-227, 233-237, 241-243, 245-247, 254, 256, 257, 260,263-265, 267-269, 272, 273, 275-277, 282, 283, 285, 286, 287, 291, 295,297, 298, 308, 309, 312, 314, 315-319, 321, 323, 324, 333-336, 340, 341,351-354, 366, 369, 373-374, 376-377, 380, 382, 386-388, 393-397, 399,401, 402, 404-409, 417-422, 424, 427-430, 436, 438-451, 453-457, 463,465, 467, 469, 471, 472, 474, 476-478, 481, 482, 485, 486, 489-492, 494,499, 511, 512, 531-535, 537, 539, 547, 548, 553, 558, 565, 567, 572-577,579, 582-584, 586-595, 597, 598, 600-605, 607-609, 612, 614-616, 618,622, 623, 625-627, 629, 630, 633, 634, 636, 642-644, 650-653, and658-666 has a K_(i) of less than 0.10 micromolar for the inhibition ofDNA-PK. For example, Compounds 661, 665, and 666 have a K_(i) of 0.001micromolar and Compound 663 has a K_(i) of 0.008 miromolar for theinhibition of DNA-PK.

Gene Editing Examples EXAMPLE 12 Materials and Methods

Methods:

Cells and Culture

Bronchial Epithelial Cells (BECs) were derived from human donorsdiagnosed with Cystic Fibrosis with a CFTR dF508/dF508 genotype.

Induced Pluripotent Stem Cells (iPSCs) were derived from human dermalfibroblasts after viral transduction with Yamanaka's reprogrammingfactors, Oct4, Sox2, KLF4 and c-Myc. Derived iPSCs were able todifferentiate into 3 germ layers and contained a normal karyotype with23 pairs of Chromosomes.

Primary human mobilized peripheral blood (mPB) CD34⁺ hematopoietic stemand progenitor cells (HSPCs) were purchased from Hemacare or AllCells.Cells were thawed, washed and resuspended in complete medium comprisedof serum free medium CellGro SCGM (CellGenix) and supplemented withcytokine mix (300 ng/mL SCF, 300 ng/mL F1t3 L, 100 ng/mL TPO, 60 ng/mlIL-3) at a density of 1-3×10⁵ cells per mL and incubated at 37° C./5%CO₂ incubator for 48 hours prior to electroporation.

DNA-PK inhibitors:

The DNA-PK inhibitor Compounds 661, 663, 665 and 666 were used for thegene editing examples. 10 mM stock solutions were made by usinganhydrous DMSO and store at −80° C.

Electroporation:

The synthetic sgRNAs used were purchased HPLC (high-performance liquidchromatography) purified from Synthego and contained chemically modifiednucleotides (2′-O-methyl 3′-phosphorothioate) at the three terminalpositions at both the 5′ and 3′ ends. The sequences of the sgRNAs withthe modified nucleotides are underlined as follows:

AAVS1 sgRNA: (SEQ ID NO: 3)5′ ACCCCACAGUGGGGCCACUAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUG CUUUU 3′.NAV 1.7 sgRNA: (SEQ ID NO: 4)5′ GGCUGAGCGUCCAUCAACCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAG UCGGUGCUUUU 3′ 

Cas9 mRNA was purchased from TriLink Biotechnologies (L-7206). Cas9 mRNAexpresses a version of the Streptococcus pyogenes SF370 Cas9 protein(CRISPR Associated Protein 9) with nuclear localization signals. spCas9mRNA also contains a CAP1 structure, a polyadenylated signal andmodified Uridines to obtain optimal expression levels in mammaliancells.

Donor ssODNs were purchase from IDT. ssODNs contain a 10 nucleotideinsertion sequence, to measure HDR events by TIDE assay, flanked by 40nucleotides of homology arms. ssODNs contain 90 nucleotides in total andPhosphorothioate modified nucleotides at the three terminal positions atboth 5′ and 3′ ends. The sequences of donor ssODNs with the underlinedPhosphorothioate modified nucleotides are indicated as follows:

AAVS1 PAM: (SEQ ID NO: 5) 5′GGGTACTTTTATCTGTCCCCTCCACCCCACAGTGGGGCCAGAATTCTCAGCTAGGGACAGGATTGGTGACAGAAAAGCCCCATCCTTAGG3′ AAVS1 Non-PAM:(SEQ ID NO: 6) 5′CCTAAGGATGGGGCTTTTCTGTCACCAATCCTGTCCCTAGCTGAGAATTCTGGCCCCACTGTGGGGTGGAGGGGACAGATAAAAGTACCC3′ NAV1.7 PAM:(SEQ ID NO: 7) 5′AGCTGTCCATTGGGGAGCATGAGGGCTGAGCGTCCATCAACTGAGAATTCCCAGGGAGACCACACCGTTGCAGTCCACAGCACTGTGCAT3′ NAV1.7 Non-PAM:(SEQ ID NO: 8) 5′ATGCACAGTGCTGTGGACTGCAACGGTGTGGTCTCCCTGGGAATTCTCAGTTGATGGACGCTCAGCCCTCATGCTCCCCAATGGACAGCT3′

All electroporations were performed on the Lonza 4D-Nucleofector™System.

For BECs, the following conditions were used for electroporation: 1.8xE5Cells, 250 ng of CAS9 mRNA, 500 ng of sgRNA and 0.66 uM of ssODN in 20ul of P4 Electroporation buffer by using program CM-138. Theelectroporated cells were transferred to a 96 well plate containing 100ul of BECs culture media supplemented with DNA-PK inhibitors or leftuntreated. Cells were incubated at 37° C. in a 5% CO₂ incubator.

For iPSCs the following conditions were used: 2.0xE5 Cells, 250 ng ofCAS9 mRNA, 500 ng of sgRNA and 0.66 uM of ssODN in 20 ul of P3Electroporation buffer by using program CA-137. The electroporated cellswere transferred to a 96 well plate containing 100 ul of mTEsR1 media(Stem Cell Technologies) supplemented with 10 uM ROCK Inhibitor Y-27632(Stem Cell Technologies) with or without DNA-PK inhibitors and thenincubated at 37° C. in a 5% CO₂ incubator.

CD34⁺ cells were electroporated two days post thaw. The followingconditions were used for electroporation: 2.0xE6 cells, 15 μg Cas9protein (Feldan), 15 μg sgRNA, 1 μM of ssODN in 100 μl of P3electroporation buffer using program CA-137. Electroporated cells weretransferred by equally dividing them into eight wells of a 24-wellplate, each well containing various concentrations of DNA-PK inhibitors.Cells were incubated at 37° C. in a 5% CO₂ incubator for two days andevaluated for cell viability and gene editing.

Lipid-mediated cell transfection:

One day prior to transfection, BECs were seeded in a 96-well plate at acell density of 1×E4 cells per well in BECs culture media. First, 0.15ul of MessengerMax (ThermoFisher, LMRNA 003) was diluted into 5 ul ofOpti-MEM and incubated for 10 min at room temperature. Meanwhile, 80 ngof Cas9 mRNA (Trilink, L-7206), 20 ng of sgRNA (Synthego) and 1 picomolof ssODN were added to 5 ul of Opti-MEM and then mixed with MessengerMAxsolution. The mixture was incubated for 5 min prior to addition to thecells. The entire solution was added to the cells in a well of 96-wellplate with 100 ul of culture media with or without DNA-PK inhibitors.Cells were incubated at 37° C. for in a 5% CO₂ incubator.

Measurement of Cell Survival Rates:

Cells were incubated with 5 ug/ml of Hoechst 33342 (Life technologies:H3570) and 0.5 ug/ml of Propidium Iodide (PI; Life technologies: P3566)in culture media for 1 h at 37 degrees. Cells were imaged to measureHoescht positive events (Live and death cells) and PI positive events(Death cells) by using a High-Content Imaging System (Moleculardevices). Relative cell survival rate was calculated as follows:[(Hoescht⁺ events−PI⁺ events) of Sample]/(Hoescht⁺ events−PI⁺ events) ofControl]*100. Control was Mock transfected cells and its cell survivalrate was set arbitrarily as 100%.

CD34⁺ HSPCs cell survival was measured using Cell Titer Glo (CTG)reagent (Promega). 100 μl of cell suspension was mixed with 100 μlcomplete CTG reagent. The chemiluminescent signal was measure using aluminometer and % of viable cells was calculated as compared to controlcells (cells not treated with the DNA-PK inhibitors).

Measurement of Gene Editing Rates:

Genomic DNA was isolated by incubating cells with 50 ul DNA Quickextractsolution (Epicentre) per well of 96 well plate for 30 min at 37° C.Cellular extract was mixed and transferred into a PCR plate and thenincubated for 6 min at 65° C. and 2 min at 98° C. PCR reactions werecarried out with 1 ul of Genomic DNA containing solution by usingAccuPrime™ Pfx DNA Polymerase (Thermofisher, 12344024). PCR conditionswere 4 min at 94° C. (1×), followed by 15 s at 94° C., 15 s at 60° C.and 1 min at 60° C. (40×). The PCR products were purified and thenSanger sequenced by GENEWIZ. The following primer pairs spanning thetarget site were used for PCR (FW, forward; RV, reverse). Primers usedby Sanger Sequencing are indicated by an asterisk (*):

(SEQ ID NO: 9) AAVS1_FW: 5′ GGACAACCCCAAAGTACCCC 3′ (SEQ ID NO: 10)AAVS1_RV*: 5′ aggatcagtgaaacgcacca 3′.  (SEQ ID NO: 11)NAV1.7_FW*: 5′ gccagtgggttcagtggtat 3′.  (SEQ ID NO: 12)NAV1.7_RV: 5′ tcagcattatccttgcattttctgt 3′. 

Each sequence chromatogram was analyzed using TIDE (Tracking of Indelsby Decomposition) software (http://tide.nki.nl) (See also Brinkman etal., Nucleic Acids Research, Volume 42, Issue 22. 16 Dec. 2014, Pagese168). Mock-electroporated samples were used as the reference sequence,and parameters were set to an indel size of 30 nt, and the decompositionwindow was set to cover the largest possible window with high-qualitytraces. Total indel (insertion and deletions) rates were obtaineddirectly from TIDE plots. HDR rates were the percentage of events withan insertion of 10 Nucleotides. NHEJ Rates were calculated as TotalIndel rate−HDR rate. GraphPad Prism 7 software was used to make Graphsand to calculate the all Statistical information.

EXAMPLE 13 DNA-PK Inhibitors Improve HDR Gene Editing Rates in BECs

FIG. 1 illustrates the design of the gene editing assyas used in theexamples below. To investigate the effect of DNA-PK inhibitors on HDRgene editing rates, BECs were electroporated with spCAS9 mRNA, NAV1.7sgRNA and NAV1.7 Non-PAM ssODN and then incubated with differentconcentrations Compound 665 or left untreated (Control). Gene editingrates were determined by using TIDE assay 72 hs after electroporation.Gene editing rates were expressed in percentages and classified as HDRand NHEJ. Cell survival rates are shown in percentages where controlcells were set as 100%.

As shown in FIG. 2, the DNA-PK inhibitor of Compound 665 improves geneediting rates in BECs. For Compound 665, the NHEJ IC50 was 0.4163 μM,the HDR EC50 was 0.4834 μM and the HDR TOP % was 76.03.

EXAMPLE 14 DNA-PK Inhibitors Improve HDR Gene Editing Rates in CD34⁺Cells

To investigate the effect of DNA-PK inhibitors on HDR gene editingrates, mPB CD34⁺ cells were electroporated with RNP (spCAS9protein+NAV1.7 sgRNA) and NAV1.7 Non-PAM ssODN. Cells were thenincubated with various concentrations of Compound 665. Gene editingrates were determined by using TIDE assay 48 h after electroporation.Gene editing rates were expressed in percentages and classified as HDRand NHEJ as shown in FIG. 3A (Donor B) and FIG. 3B (Donor C). Cellsurvival rates are shown in percentages where control cells were set as100%.

As shown in FIGS. 3A and 3B, the DNA-PK inhibitor of Compound 665improves gene editing rates in CD34⁺ cells. EC50 values of HDR and Indelformation for Donor B were 0.28 μM and 0.36 μM, respectively.

EXAMPLE 15 DNA-PK Inhibitors Improve HDR Gene Editing Rates in iPSCs

To investigate the effect of DNA-PK inhibitors on HDR gene editingrates, iPSCs were electroporated with spCAS9 mRNA, AAVS1 sgRNA and AAVS1PAM ssODN and then incubated with different concentrations of Compound665 or left untreated (Control). Gene editing rates were determined byusing TIDE assay 72 hs after electroporation. Gene editing rates wereexpressed in percentages and classified as HDR and NHEJ. Cell survivalrates are shown in percentages where control cells were set as 100%.

As shown in FIG. 4, the DNA-PK inhibitor of Compound 665 improves geneediting rates in iPSCs. For Compound 665, the NHEJ IC50 was 1.274 μM,the HDR EC50 was 0.9337 μM and the HDR TOP % was 26.27.

EXAMPLE 16 Determination of Gene Editing Kinetics at ECmax

To investigate the gene editing kinetics at EC max, BECs wereelectroporated with spCAS9 mRNA, AAVS1 sgRNA and AAVS1 PAM ssODN andthen incubated at different times with 10 μM Compound 665 or leftuntreated (Control). Gene editing rates were determined by using TIDEassay and expressed in percentages of HDR and NHEJ. 10 μM is the MaximumEnhance Concentration (ECmax) of Compound 665.

FIG. 5 shows that there is a tight inverse correlation between HDR andNHEJ events.

EXAMPLE 17 Determination of Gene Editing Kinetics at EC50

BECs were electroporated with spCAS9 mRNA, AAVS1 sgRNA and AAVS1 PAMssODN and then incubated at different times with 0.7 μM Compound 665 orleft untreated (Control). Gene editing rates were determined by usingTIDE assay and expressed in percentages of HDR and NHEJ. 0.7 μM is theEnhance Concentration 50 (EC50) of Compound 665. FIG. 6 illustrates thetime course of DNA-PK inhibition on HDR and NHEJ in BECs.

EXAMPLE 18 DNA-PK Inhibitors Improve HDR Rates when Gene EditingComponents were Delivered by Lipid-Mediated Transfection in BECs

To investigate the effects of lipid-mediated transfection, BECs weretransfected with spCAS9 mRNA, AAVS1 sgRNA and AAVS1 PAM ssODN and thenincubated with different concentration of Compound 665 or left untreated(Control). Gene editing rates were determined by using TIDE assay 72 hsafter transfection. FIG. 7 shows increasing HDR efficiency rates withincreasing concentrations of Compound 665 delivered by lipid-basedtransfection.

Summary Table: DNA-PK Inhibitor of Compound 665 Improves HDR Driven GeneEditing Compound 665 AAVS1 NaV1.7 Cells HDR EC50 (μM) and Max % BECs0.70 μM 0.48 μM 72% 76% iPSCs 0.93 μM N.D. 26% N.D. CD34⁺'s Donor A 0.38μM 0.29 μM 84% 81% Donor B N.D. 0.28 μM N.D. 86% Donor C 0.32 μM 0.34 μM92% 68%

EXAMPLE 19

mPB CD34⁺ cells were electroporated with RNP (spCAS9 protein+NAV1.7sgRNA) and NAV1.7 Non-PAM ssODN. Cells were then incubated with variousconcentrations of Compounds 661, 663 and 666. Experiments were done asdescribed in the methods above, except lower concentrations of sgRNA (5μg) and ssODN (0.2 μM) were used. Gene editing rates were determined byusing TIDE assay 48 h after electroporation. Gene editing rates wereexpressed in percentages and classified as HDR and NHEJ. The results foreach Compound tested in two separate donors are shown below.

Compound 661

Compound 661 Compound Donor A Donor D Conc % % % % % % (μM) HDR IndelsWT Viability HDR Indels WT Viability 10.00 28 47 18.9 6 13.4 64.2 11.111 3.33 32.2 57.8 5.1 47 11.5 76.7 6.4 75 1.11 31.7 59.4 4.2 60 10.877.5 6 62 0 11.9 77.4 3.3 100 3.2 76.2 7.5 100

Compound 663

Compound 663 Compound Donor A Donor D Conc % % % % % % (μM) HDR IndelsWT Viability HDR Indels WT Viability 10.00 24.3 46.5 24.1 10 13.9 76.2 112 3.33 37 11.3 81.8 1.9 64 1.11 35.7 55.3 4.4 65 8.5 81.3 5.4 83 0 11.977.4 3.3 100 3.2 76.2 7.5 100

Compound 666

Compound 666 Compound Donor A Donor D Conc % % % % % % (μM) HDR IndelsWT Viability HDR Indels WT Viability 10.00 33.6 52 9.7 36 13.7 69 11.834 3.33 32.4 56.5 5.8 42 14.3 73.8 5.3 69 1.11 31.5 58.8 3.5 65 11 77.56.2 76 0 11.9 77.4 3.3 100 3.2 76.2 7.5 100

Summary Table for Compounds 661, 663, 666 Conc. Donor A Donor D (μM) %HDR % NHEJ % HDR % NHEJ Compound 666  0 11.9 77.4  3.2 76.2  0.37 23.267.3  5.5 79.2  1.11 31.5 58.8 11 77.5 10 33.6 52 13.7 69 Compound 661 0 11.9 77.4  3.2 76.2  0.37 21.8 66.2  6.9 78.1  1.11 31.7 59.4 10.877.5 10 28 47 13.4 64.2 Compound 663  0 11.9 77.4  3.2 76.2  0.37 24.664.8  8.9 79.4  1.11 35.7 55.3  8.5 81.3 10 24.3 46.5 13.9 76.2

EXAMPLE 20 DNA-PK Inhibitors Improve HDR Gene Editing Rates in CD34⁺Cells

To investigate the effect of DNA-PK inhibitors on HDR gene editingrates, CD34⁺ cells were electroporated with spCAS9 mRNA, AAVS1 sgRNA andAAVS1 Non-PAM ssODN and then incubated with different concentrations ofCompounds 666, 661, 663 or left untreated (Control). Gene editing rateswere determined by using TIDE assay 72 hs after electroporation. Geneediting rates were expressed in percentages and classified as HDR andNHEJ. Cell survival rates are shown in percentages where control cellswere set as 100%.

As shown, the DNA-PK inhibitors of Compounds 666 (FIG. 8), 661 (FIG. 9)and 663 (FIG. 10) improve HDR rates in CD34⁺ cells.

EXAMPLE 21 Precise Gene Editing by HDR Mediated by AAV Donors,CRISPR-Cas9 and Selective DNA-PK Inhibitors

FIG. 11 illustrates the design of the gene editing assay used in thisexample.

Cells

Lung Progenitor Cells (LPCs) were derived from human lung donorsdiagnosed with Cystic Fibrosis. LPC donor ID 14071 and 14335 contain theCFTR genotypes dF508/dF508 and dF508/G542X, respectively.

CRISPR-Cas9 Gene Editing Reagents

Synthetic sgRNAs were purchased from Synthego. gRNAs were HPLC(high-performance liquid chromatography) purified and contain chemicallymodified nucleotides (2′-O-methyl 3′-phosphorothioate) at the threeterminal positions at both the 5′ and 3′ ends. gRNAs contain a 22nucleotides long spacer sequence to promote specific binding to targetsite and 80 nucleotides long scaffold sequence that allows binding tosaCAS9 protein. Complete gRNA sequences are shown in Table 3.

saCas9 mRNA was synthetized by TriLink Biotechnologies. saCas9 mRNAexpresses a Staphylococcus aureus Cas9 (Uniprot entry code J7RUA5) withSV40 and NucleoPlasmine nuclear localization signals. saCas9 mRNA alsocontains a CAP1 structure and a polyadenylated signal to obtain optimalexpression levels in mammalian cells. saCAS9 mRNA was HPLC purified.

AAV donor constructs design and AAV transductions.

AAV donor constructs contain 500 nucleotide long sequences of left andright homology arms relative to gRNA cut site and a unique HDR footprintinsertion of 10 nucleotides. AAV donor preparations were made by usingAAV6 serotype, purified and titrated by Vector Biolabs. AAV titrationwas reported as viral genomes per ml. AAV transduction were done byadding AAV6 vector into cells at specified vector genome copies per cellduring 36 h at 37° C.

Electroporation

Electroporations were performed by using the Lonza 4D-Nucleofector™System coupled to 96-well shuttle system. 1.8xE5 LPC cells wereresuspended in 20 ul of P4 Electroporation buffer Lonza (V4SP-4096). 20ul of cell mixture was combined with 2 ul of CRISPR-Cas9 reagent mixcontaining 1 ug of saCAS9 mRNA and 1 ug of gRNA. 20 ul of cell andCRISPR-Cas9 mixture was transferred into one well of a 96-wellelectroporation plate. Cells were electroporated by using programCM-138. A fraction of electroporated LPC cells were transferred into awell of 384-well plate. Cells were transduced with AAV and DNA-PKinhibitor or left untreated (Control) during 36 hs in a 5% CO2incubator. Genomic DNA was isolated after 72 h.

Genomic DNA Isolation.

Genomic DNA was isolated by incubating cells during 30 min at 37° C.with 50 ul and 15 ul of DNA Quickextract solution (Epicentre) per wellof 96-well and 384-well plate, respectively. Cellular extract was mixedand transferred into a 96-well PCR plate and then incubated for 6 min at65° C. and 2 min at 98° C. Genomic DNA was immediately use in downstreamapplications or it was store at 4° C.

Measurement of INDEL Rates

Phire Green Hot Start II PCR Master Mix (F126 L, Thermo Scientific) wasused to amplify DNA fragment corresponding to target genes. PCRreactions were carried out following manufacturer instructions. Inbrief, 1.25 ul of genomic DNA solution was combined with 23.5 ul ofPhire Green Hot Start II PCR Master Mix and the corresponding targetgenes forward and reverse primers (Table 4). One of the primers bindsoutside of AAV donor sequence to avoid amplification of AAV donor. PCRreactions were performed with the following thermal cycling protocol: 1)98° C. 30 s; 2), 98° C. 5 s; 3), 62° C. 5 s; 4) 72° C. 20 s repeat steps2 to 4 30 times; 5) 72° C. 4 min The PCR products were enzymaticallypurified. DNA samples were Sanger sequenced by using sequencing primersas shown in Table 4. Each sequencing chromatogram was analyzed usingTIDE software against reference sequences (described above). Referencessequences were obtained from mock-electroporated samples. Tideparameters were set to cover an indel spectrum of +/−30 nucleotides ofgRNA cut site and the decomposition window was set to cover the largestpossible window with high-quality traces. Total indel (insertion anddeletions) rates were obtained directly from TIDE plots. GraphPad Prism7 software was used to make Graphs and to calculate the all Statisticalinformation.

TABLE 3  sgRNAs sequences Target sgRNA VEGFAAUUCCCUCUUUAGCCAGAGCGUUUUAGUACUCUGGAAACAGAAUCUACUAAAACAAGGCAAAAUGCCGUGUUUAUCUCGUCAACUUGUUGGCGAGAUUUU (SEQ ID NO: 13) EMX1CAACCACAAACCCACGAGGGGUUUUAGUACUCUGGAAACAGAAUCUACUAAAACAAGGCAAAAUGCCGUGUUUAUCUCGUCAACUUGUUGGCGAGAUUUU (SEQ ID NO: 14) FANCFCAAGGCCCGGCGCACGGUGGGUUUUAGUACUCUGGAAACAGAAUCUACUAAAACAAGGCAAAAUGCCGUGUUUAUCUCGUCAACUUGUUGGCGAGAUUUU (SEQ ID NO: 15) RUNXAAAGAGAGAUGUAGGGCUAGGUUUUAGUACUCUGGAAACAGAAUCUACUAAAACAAGGCAAAAUGCCGUGUUUAUCUCGUCAACUUGUUGGCGAGAUUUU (SEQ ID NO: 16)

Note: Each sgRNA contain a unique 22 nucleotides spacer sequence (Bold)follow by a common 80 nucleotides scaffold sequence.

TABLE 4  PCR and Sequencing primers PCR Forward PCR Reverse Sequencing Gene primer Primer primer VEGFA AGACGTTCCTT AGGAGGGAGCA ATTCCCTCTTTAGAGTGCTGGC GGAAAGTGA CCAGAGC (SEQ ID NO: 17) (SEQ ID NO: 18)(SEQ ID NO: 19) EMX1 TGGCTGTCCAG GGCCTGTCCTC CAACCACAAACCC GCACTGCTCCCTCAAG ACGAGGG (SEQ ID NO: 20) (SEQ ID NO: 21) (SEQ ID NO: 22) FANCFACACGGATAAA ACACGGATAAA CAAGGCCCGGCGC GACGCTGGG GACGCTGGG ACGGTGG(SEQ ID NO: 23) (SEQ ID NO: 24) (SEQ ID NO: 25) RUNX1 AACCCAGCATATCTGTGCATGT AAAGAGAGATGTA GTGGTCAGC GCCTGCTAA GGGCTAG (SEQ ID NO: 26)(SEQ ID NO: 27) (SEQ ID NO: 28)

LPCs were electroporated with saCAS9 mRNA together a set of sgRNA andAAV donors to target Fanconi anemia complementation group F (FANCF),runt-related transcription factor 1 (RUNX1), Empty Spiracles Homeobox 1(EMX1) and Vascular endothelial growth factor A (VEGFA) genes. Cellswere incubated with DNA-PK inhibitor Compound 665 or left untreated(Control). Total Insertion and Deletions (INDELs) rates were determinedby using TIDE assay 72 h after electroporation. Gene editing rates wereexpressed in percentages and classified as HDR (Bars) and NHEJ(Circles). HDR events were the amount of sequences with 10 nucleotidesinsertion. NHEJ rates were calculated by using the following formula:NHEJ=Total INDEL events−HDR events. Results are shown in FIG. 12.

Summary:

Results of the addition of DNA-PK inhibitors to different cell types andloci show significant enhancement of HDR across cell types and lociEnhancement of HDR gene editing has been shown in multiple cell typesincluding BECs, iPSCs, CD34⁺ HPSCs (3 separate donors) Enhancement ofHDR gene editing has been shown in multiple loci. Experimental resultshave also shown that lipid based and electroporation delivery iseffective. Electroporation examples include BECs, iPSCs, CD34⁺ HPSCs andeffect delivery using a lipid-based delivery system in BECs. A tightinverse correlation between HDR and NHEJ events has been observed acrossloci, experimental conditions and cell types.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

1. A method of repairing a DNA break in one or more target genomicregions via a homology directed repair (HDR) pathway, comprising:administering to one or more cells that comprise one or more targetgenomic regions, a genome editing system and a compound of formula(III-E-1) or (III-E-2), or a pharmaceutically acceptable salt thereof,

wherein: X is O or NR; wherein R is H or C₁-C₄ alkyl; Y is O, or NR;wherein R is H or C₁-C₄ alkyl; R³ is hydrogen, C₁₋₄ alkyl, or OC₁₋₂alkyl; R¹ is a 6-membered heteroaromatic ring containing one or twonitrogen atoms wherein the heteroaromatic ring may be substituted by 0,1, 2 or 3 substituents R² independently selected from the groupconsisting of halo, CN, C₁-C₄-alkyl, C₁-C₄-haloalkyl, C₃-C₄-cycloalkyl,OR⁶, C(═O)OR⁶, C(═O)NR⁷R⁶, and NR⁴R⁵; wherein each C₁-C₄-alkyl andC₁-C₄-haloalkyl is substituted by 0, 1, or 2 OR⁶ groups, each R⁶ and R⁷is independently H, C₁-C₄ alkyl or C₁-C₄-haloalkyl, each R⁴ and R⁵ isindependently H, C₁C4 alkyl, or C(═O)C₁-C₄ alkyl; or R⁴ and R⁵ togetherwith the N atom to which they are attached form a heterocyclic ringcomprising 0 or 1 additional O or N atom wherein said heterocyclic ringmay be substituted by C₁-C₄ alkyl or OR⁶; and Ring B is selected fromthe group consisting of:

wherein W is N or CR⁷; and Z is O or S; wherein R⁷ is H or C₁-C₄ alkyl,wherein the genome editing system interacts with a nucleic acid(s) ofthe target genomic regions, resulting in a DNA break, and wherein theDNA break is repaired at least in part via a HDR pathway.
 2. (canceled)3. A method of inhibiting or suppressing repair of a DNA break in one ormore target genomic regions via a non-homologous end joining (NHEJ)pathway, comprising: administering to one or more cells that compriseone or more target genomic regions, a genome editing system and acompound of formula (III-E-1) or (III-E-2), or a pharmaceuticallyacceptable salt thereof,

wherein: X is O or NR; wherein R is H or C₁-C₄ alkyl; Y is O, or NR;wherein R is H or C₁-C₄ alkyl; R³ is hydrogen, C₁₋₄ alkyl, or OC₁₋₂alkyl; R¹ is a 6-membered heteroaromatic ring containing one or twonitrogen atoms wherein the heteroaromatic ring may be substituted by 0,1, 2 or 3 substituents R² independently selected from the groupconsisting of halo, CN, C₁-C₄-alkyl, C₁-C₄-haloalkyl, C₃-C₄-cycloalkyl,OR⁶, C(═O)OR⁶, C(═O)NR⁷R⁶, and NR⁴R⁵; wherein each C₁-C₄-alkyl andC₁-C₄-haloalkyl is substituted by 0, 1, or 2 OR⁶ groups, each R⁶ and R⁷is independently H, C₁-C₄ alkyl or C₁-C₄-haloalkyl, each R⁴ and R⁵ isindependently H, C₁C₄ alkyl, or C(═O)C₁-C₄ alkyl; or R⁴ and R⁵ togetherwith the N atom to which they are attached form a heterocyclic ringcomprising 0 or 1 additional O or N atom wherein said heterocyclic ringmay be substituted by C₁-C₄ alkyl or OR⁶; and Ring B is selected fromthe group consisting of:

wherein W is N or CR⁷; and Z is O or S; wherein R⁷ is H or C₁-C₄ alkyl,wherein the genome editing system interacts with a nucleic acid(s) ofthe one or more target genomic regions, resulting in a DNA break, andwherein repair of the DNA break via a NHEJ pathway is inhibited orsuppressed.
 4. A method of modifying expression of one or more genes orproteins comprising: administering to one or more cells that compriseone or more target genomic regions, a genome editing system and acompound of formula (III-E-1) or (III-E-2), or a pharmaceuticallyacceptable salt thereof,

wherein: X is O or NR; wherein R is H or C₁-C₄ alkyl; Y is O, or NR;wherein R is H or C₁-C₄ alkyl; R³ is hydrogen, C₁₋₄ alkyl, or OC₁₋₂alkyl; R¹ is a 6-membered heteroaromatic ring containing one or twonitrogen atoms wherein the heteroaromatic ring may be substituted by 0,1, 2 or 3 substituents R² independently selected from the groupconsisting of halo, CN, C₁-C₄-alkyl, C₁-C₄-haloalkyl, C₃-C₄-cycloalkyl,OR⁶, C(═O)OR⁶, C(═O)NR⁷R⁶, and NR⁴R⁵; wherein each C₁-C₄-alkyl andC₁-C₄-haloalkyl is substituted by 0, 1, or 2 OR⁶ groups, each R⁶ and R⁷is independently H, C₁-C₄ alkyl or C₁-C₄-haloalkyl, each R⁴ and R⁵ isindependently H, C₁C₄ alkyl, or C(═O)C₁-C₄ alkyl; or R⁴ and R⁵ togetherwith the N atom to which they are attached form a heterocyclic ringcomprising 0 or 1 additional O or N atom wherein said heterocyclic ringmay be substituted by C₁-C₄ alkyl or OR⁶; and Ring B is selected fromthe grout) consisting of:

wherein W is N or CR⁷; and Z is O or S; wherein R⁷ is H or C₁-C₄ alkyl,wherein the genome editing system interacts with a nucleic acid(s) ofthe one or more target genomic regions of a target gene(s), resulting inediting the one or more target genomic regions and wherein the editmodifies expression of a downstream gene(s) and/or protein(s) associatedwith the target gene(s).
 5. The method of claim 1, wherein X is O or NR;wherein R is H or C₁-C₄ alkyl; Y is O, or NR; wherein R is H or C₁-C₄alkyl; R³ is hydrogen, C₁₋₄ alkyl, or OC₁₋₂ alkyl; R¹ is a 6-memberedheteroaromatic ring containing one or two nitrogen atoms wherein theheteroaromatic ring may be substituted by 0, 1, or 2 substituents R²independently selected from the group consisting of C₁-C₄-alkyl,C₁-C₄-haloalkyl, C(═O)NHR⁶, and NR⁴R⁵; wherein R⁶ is C₁-C₄ alkyl, eachR⁴ and R⁵ is independently H, C₁-C₄ alkyl, or C(═O)C₁-C₄ alkyl; or R⁴and R⁵ together with the N atom to which they are attached form aheterocyclic ring comprising 0 or 1 additional N atom wherein saidheterocyclic ring may be substituted by C₁-C₄ alkyl; and Ring B isselected from the group consisting of:

wherein W is N or CR⁷; and Z is O or S; wherein R⁷ is H or C₁-C₄ alkyl.6. The method of claim 1, wherein X is O or NH, Y is O or NH, and R³ ishydrogen.
 7. The method of claim 1, wherein X is O or NH, Y is O or NH,R³ is hydrogen and R¹ is a pyrimidine ring which is substituted by 0, 1,or 2 substituents R² independently selected from the group consisting ofC₁-C₄-alkyl, C₁-C₄-haloalkyl, C(═O)NHR⁶, and NR⁴R⁵.
 8. The method ofclaim 1, wherein X is O or NH, Y is O or NH, R³ is hydrogen and R¹ is apyrimidine ring which is substituted by 0, 1, or 2 substituents R²independently selected from the group consisting of C₁-C₄-alkyl,C₁-C₄-haloalkyl, C(═O)NHR⁶, and NR⁴R⁵; Ring B is selected from the groupconsisting of:

W is N or CR⁷; and Z is O or S; wherein R⁷ is H or C₁-C₄-alkyl.
 9. Themethod of claim 1, X is O or NH, Y is O or NH, R³ is hydrogen and R¹ isa pyrimidine ring which is substituted by 0, 1, or 2 substituents R²independently selected from the group consisting of C₁-C₄ alkyl,C₁-C₄-haloalkyl, C(═O)NHR⁶, and NR⁴R⁵; Ring B is

W is N or CR⁷; and Z is O or S; wherein R⁷ is H or C₁C₄ alkyl.
 10. Themethod of claim 1, wherein X is O or NH, Y is O or NH, R³ is hydrogenand R¹ is a pyrimidine ring which is substituted by 0, 1, or 2substituents R² independently selected from the group consisting ofC₁-C₄-alkyl and C(═O)NHR⁶; Ring B is

W is N; and Z is O or S.
 11. The method of claim 1, wherein X is O orNH, Y is O or NH, R³ is hydrogen and R¹ is a pyrimidine ring which issubstituted by one substituent R² selected from the group consisting ofC₁-C₂-alkyl and C(═O)NH C₁-C₂-alkyl; Ring B is

W is N; and Z is O or S.
 12. The method of claim 1, wherein the compoundis represented by one one of the following formulae or apharmaceutically acceptable salt thereof:


13. The method of claim 3, wherein the DNA break comprises a DNA doublestrand break (DSB).
 14. The method of claim 1, wherein the efficiency ofediting the target genomic regions in the one or more cells is increasedas compared to that in otherwise identical cell or cells but without thecompound.
 15. The method of 5, wherein the efficiency of the repair ofthe DNA break at the target genomic regions in the one or more cells viaa HDR pathway is increased as compared to that in otherwise identicalcell or cells but without the compound.
 16. The method of claim 3,wherein the efficiency of inhibiting or suppressing the repair of theDNA break at the target genomic regions in the one or more cells via aNHEJ pathway is increased as compared to that in otherwise identicalcell or cells but without the compound.
 17. (canceled)
 18. The method ofclaim 14, wherein said efficiency is measured by frequency of targetedpolynucleotide integration or targeted mutagenesis. 19-20. (canceled)21. The method of claim 4, wherein the expression of a downstream gene(s) and/or protein(s) associated with the target gene(s) is decreased orincreased as compared to the baseline expression level in the one ormore cells prior to the administration. 22-24. (canceled)
 25. The methodof claim 4, wherein the expression of a downstream gene(s) and/orprotein(s) associated with the target gene(s) is substantiallyeliminated in the one or more cells.
 26. (canceled)
 27. The method ofclaim 1, wherein the one or more cells that are administered orcontacted with said compound have increased survival in comparison toone or more cells that have not been administered or contacted with saidcompound.
 28. The method of claim 1, wherein the genome editing systemand the compound are administered into the one or more cellssimultaneously or sequentially.
 29. (canceled)
 30. The method of claim29, wherein the genome editing system is administered into the one ormore cells prior to or after administration of the compound. 31-32.(canceled)
 33. The method of claim 1, wherein the one or more cells arein vivo cells within an organism or ex vivo cells from an organism.34-36. (canceled)
 37. The method of claim 1, wherein the genome editingsystem and the compound are administered via same or different route.38. (canceled)
 39. The method of claim 38, wherein the genome editingsystem is administered intravenously and the compound is administeredorally.
 40. The method of claim 1, wherein the genome editing system isselected from a meganuclease based system, a zinc finger nuclease (ZFN)based system, a Transcription Activator-Like Effector-based Nuclease(TALEN) system, a CRISPR-based system, or a NgAgo-based system.
 41. Themethod of claim 40, wherein genome editing system is a CRISPR-basedsystem.
 42. The method of claim 41, wherein the CRISPR-based system is aCRISPR-Cas system or a CRISPR-Cpf system.
 43. The method of claim 42,wherein the CRISPR-based system is a CRISPR-Cas system and wherein theCRISPR-Cas system comprises: (a) at least one guide RNA elementcomprising: (i) a targeter RNA comprising a nucleotide sequencesubstantially complementary to a nucleotide sequence at the one or moretarget genomic regions or a nucleic acid comprising a nucleotidesequence(s) encoding the targeter RNA; (ii) and an activator RNAcomprising a nucleotide sequence that is capable of hybridizing with thetargeter RNA or a nucleic acid comprising a nucleotide sequence(s)encoding the activator RNA; and (b) a Cas protein element comprising aCas protein or a nucleic acid comprising a nucleotide sequence(s)encoding the Cas protein.
 44. The method of claim 43, wherein saidtargeter RNA and activator RNA are fused as a single molecule.
 45. Themethod of claim 43 wherein the Cas protein is a Type-II Cas9 protein.46. The method of claim 45, wherein the Cas9 protein is a SaCas9,SpCas9, SpCas9n, Cas9-HF, Cas9-H840A, FokI-dCas9, or D10A nickase, orany combinations thereof.
 47. The method of claim 42, wherein theCRISPR-based system is a CRISPR-Cpf system and wherein the CRISPR-Cpfsystem comprises: (a) at least one guide RNA element or a nucleic acidcomprising a nucleotide sequence(s) encoding the guide RNA element, theguide RNA comprising a targeter RNA that comprises a nucleotide sequencesubstantially complementary to a nucleotide sequence at the one or moretarget genomic regions; and (b) a Cpf protein element comprising a Cpfprotein or a nucleic acid comprising a nucleotide sequence encoding theCpf protein.
 48. The method of claim 1, wherein the genome editingsystem is delivered by one or more vectors, by synthetic RNA or by ananoformulation. 49-52. (canceled)
 53. A kit or composition for editingone or more target genomic regions, comprising: a genome editing system;and a compound of formula (III-E-1) or (III-E-2), or a pharmaceuticallyacceptable salt thereof,

wherein: X is O or NR; wherein R is H or C₁-C₄ alkyl; Y is O, or NR;wherein R is H or C₁-C₄ alkyl; R³ is hydrogen, C₁₋₄ alkyl, or OC₁₋₂alkyl; R¹ is a 6-membered heteroaromatic ring containing one or twonitrogen atoms wherein the heteroaromatic ring may be substituted by 0,1, 2 or 3 substituents R² independently selected from the groupconsisting of halo, CN, C₁-C₄-alkyl, C₁-C₄-haloalkyl, C₃-C₄-cycloalkyl,OR⁶, C(═O)0R⁶, C(═O)NR⁷R⁶, and NR⁴R⁵; wherein each C₁-C₄-alkyl andC₁-C₄-haloalkyl is substituted by 0, 1, or 2 OR⁶ groups, each R⁶ and R⁷is independently H, C₁-C₄ alkyl or C₁-C₄-haloalkyl, each R⁴ and R⁵ isindependently H, C₁-C₄ alkyl, or C(═O)C₁-C₄ alkyl; or R⁴ and R⁵ togetherwith the N atom to which they are attached form a heterocyclic ringcomprising 0 or 1 additional O or N atom wherein said heterocyclic ringmay be substituted by C₁-C₄ alkyl or OR⁶; and Ring B is selected fromthe group consisting of:

wherein W is N or CR⁷; and Z is O or S; wherein R⁷ is H or C₁-C₄ alkyl.54. The kit or composition of claim 53, wherein R¹ is a 6-memberedheteroaromatic ring containing one or two nitrogen atoms wherein theheteroaromatic ring may be substituted by 0, 1, or 2 substituents R²independently selected from the group consisting of C₁-C₄-alkyl,C₁-C₄-haloalkyl, C(═O)NHR⁶, and NR⁴R⁵; wherein R⁶ is C₁-C₄ alkyl, eachR⁴ and R⁵ is independently H, C₁-C₄ alkyl, or C(═O)C₁-C₄ alkyl; or R⁴and R⁵ together with the N atom to which they are attached form aheterocyclic ring comprising 0 or 1 additional N atom wherein saidheterocyclic ring may be substituted by C₁-C₄ alkyl.
 55. The kit orcomposition of claim 53, wherein the genome editing system is ameganuclease based system, a zinc finger nuclease (ZFN) based system, aTranscription Activator-Like Effector-based Nuclease (TALEN) system, aCRISPR-based system, or NgAgo-based system.
 56. The kit or compositionof claim 55, wherein genome editing system is a CRISPR-based system. 57.The kit or composition of claim 56, wherein the CRISPR-based system is aCRISPR-Cas system or a CRISPR-Cpf system.
 58. The kit or composition ofclaim 57, wherein the CRISPR-based system is a CRISPR-Cas system andwherein the CRISPR-Cas system comprises: (a) at least one guide RNAelement comprising: (i) a targeter RNA comprising a nucleotide sequencesubstantially complementary to a nucleotide sequence at the one or moretarget genomic regions or a nucleic acid comprising a nucleotidesequence(s) encoding the targeter RNA; (ii) and an activator RNAcomprising a nucleotide sequence that is capable of hybridizing with thetargeter RNA, or a nucleic acid comprising a nucleotide sequence(s)encoding the activator RNA; and (b) a Cas protein element comprising aCas protein or a nucleic acid comprising a nucleotide sequence(s)encoding the Cas protein.
 59. The kit or composition of claim 57,wherein the Cas protein is a Type-II Cas9 protein.
 60. The kit orcomposition of claim 57, wherein the Cas9 protein is a SaCas9, SpCas9,SpCas9n, Cas9-HF, Cas9-H840A, FokI-dCas9, or D10A nickase, or anycombination thereof.
 61. The kit or composition of claim 57, wherein theCRISPR-based system is a CRISPR-Cpf system, and wherein the CRISPR-Cpfsystem comprises: (a) a targeter RNA comprising a nucleotide sequencesubstantially complementary to a nucleotide sequence at the one or moretarget genomic regions, or a nucleic acid comprising a nucleotidesequence(s) encoding the targeter RNA; and (b) a Cpf protein elementcomprising a Cpf protein or a nucleic acid comprising a nucleotidesequence(s) encoding the Cpf protein.
 62. The kit or composition ofclaim 53, wherein the genome editing system is included or packaged inone or more vectors. 63-64. (canceled)
 65. A compound represented by anyone of the following formulae or a pharmaceutically acceptable saltthereof:


66. A method of repairing, or inhibiting or suppressing repair of, a DNAbreak in one or more target genomic regions via a homology directedrepair (HDR) pathway, or modifying expression of one or more genes orproteins comprising: administering to one or more cells that compriseone or more target genomic regions, a genome editing system and acompound of claim 65.